Bio-engineered hyper-functional “super” helicases

ABSTRACT

Conformationally-constrained helicases having improved activity and strength are provided. Methods of making conformationally-constrained helicases having improved activity and strength are provided. Methods of using conformationally-constrained helicases having improved activity and strength are provided. The present invention is based on the discovery of novel modified helicases that show dramatically enhanced helicase activity and increased strength as compared to unmodified helicases. As described further herein, it has been surprisingly discovered that, by controlling the conformation of certain subdomains such that the helicase remains in a closed form (e.g., by covalently crosslinking the 2B domain to the 1A domain or the 1B domain in a Rep helicase), a highly active and strong form of the helicase is achieved.

RELATED APPLICATIONS

This application is a 35 U.S.C. § 371 filing of International PatentApplication No. PCT/US2015/060693, filed Nov. 13, 2015, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 62/079,183,filed Nov. 13, 2014, the disclosures of which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under GM065367 awardedby the National institutes of Health. The United States Government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods fixhelicase-mediated DNA unwinding activity.

BACKGROUND

A traditional definition of a helicase is an enzyme that catalyzes thereaction of separating/unzipping/unwinding the helical structure ofnucleic acid duplexes (DNA, RNA or hybrids) into single-strandedcomponents, using nucleoside triphosphate (NTP) hydrolysis as the energysource (such as ATP). However, it should be noted that not all helicasesfit this definition anymore. A more general definition is that they aremotor proteins that move along the single-stranded or double strandednucleic acids (usually in a certain direction, 3′ to 5′ or 5 to 3, orboth), i.e. translocases, that can or cannot unwind the duplexed nucleicacid encountered. In addition, some helicases simply bind and “melt” theduplexed nucleic acid structure without an apparent translocaseactivity.

Helicases exist in all living organisms and function in all aspects ofnucleic acid metabolism. Helicases are classified based on the aminoacid sequences, directionality, oligomerization state and nucleic-acidtype and structure preferences. The most common classification methodwas developed based on the presence of certain amino acid sequences,called motifs. According to this classification helicases are dividedinto 6 super families: SF1, SF2, SF3, SF4, SF5 and SF6. SF1 and SF2helicases do not form a ring structure around the nucleic acid, whereasSF3 to SF6 do. Superfamily classification is not dependent on theclassical taxonomy.

DNA helicases are responsible for catalyzing the unwinding ofdouble-stranded DNA (dsDNA) molecules to their respectivesingle-stranded nucleic acid (ssDNA) forms. Although structural andbiochemical studies have shown how various helicases can translocate onssDNA directionally, consuming one ATP per nucleotide, the mechanism ofnucleic acid unwinding and how the unwinding activity is regulatedremains unclear and controversial (T. M. Lohman, E. J. Tomko, C. G. Wu,“Non-hexameric DNA helicases and translocases: mechanisms andregulation,” Nat Rev Mol Cell Biol 9:391-401 (2008)). Since helicasescan potentially unwind all nucleic acids encountered, understanding howtheir unwinding activities are regulated can lead to harnessing helicasefunctions for biotechnology applications.

BRIEF SUMMARY OF THE INVENTION

The present invention is based on the discovery of novel modifiedhelicases that show dramatically enhanced helicase activity andincreased strength as compared to unmodified helicases. As describedfurther herein, it has been surprisingly discovered that, by controllingthe conformation of certain subdomains such that the helicase remains ina closed form (e.g., by covalently crosslinking the 2B domain to the 1Adomain or the 1B domain in a Rep helicase), a highly active and strongform of the helicase is achieved.

In one aspect, a composition for catalyzing an unwinding reaction ondouble-stranded DNA is provided that includes aconformationally-constrained helicase.

In another aspect, a method of catalyzing an unwinding reaction of adouble-stranded DNA is provided. The method includes the step ofcontacting the double-stranded DNA with a conformationally-constrainedhelicase in the presence of ATP.

In another aspect, an isolated nucleic acid that encodes a helicasepolypeptide having the capability to be constrained in a conformation byan intramolecular crosslinking agent is provided.

In another aspect, a modified helicase comprising a first subdomainhaving a first amino acid and a second subdomain having a second aminoacid is provided. Said first amino acid is at least about 30 Å from saidsecond amino acid when the helicase is in an inactive conformation, andsaid first amino acid is less than about 20 Å from said second aminoacid when the helicase is in an active conformation. A side chain of thefirst amino acid is covalently crosslinked to a side chain of the secondamino acid with a linker to form an active, conformationally-constrainedhelicase.

In certain exemplary embodiments, the modified helicase is a SuperFamily 1 (SF1) helicase (e.g., an SF1A or an SF1B helicase) or a SuperFamily 2 (SF2) helicase.

In certain exemplary embodiments, the first amino acid is less thanabout 20 Å, about 19 Å, about 18 Å, about 17 Å, about 16 Å, about 15 Å,about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Åfrom the second amino acid when the helicase is in an activeconformation.

In certain exemplary embodiments, the first amino acid is at least about30 Å, about 40 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å, about70 Å, about 75 Å, about 80 Å or about 85 Å from the second amino acidwhen the helicase is in an inactive conformation.

In certain exemplary embodiments, the helicase is selected from thegroup consisting of a Rep helicase (e.g., from E. coli), a UvrD helicase(e.g., from E. coli) and a PcrA helicase (e.g., from B.stearothermophilus).

In certain exemplary embodiments, the first amino acid is at any one ofpositions 84-116 or 178-196 of the modified helicase amino acidsequence, and the helicase is a Rep, PcrA or UvrD helicase, or homologthereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 92-116 or 178-196 of the modified helicase amino acidsequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 84-108 or 169-187 of the modified helicase amino acidsequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 90-114 or 175-193 of the modified helicase amino acidsequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid at position 178of the modified helicase amino acid sequence, and the helicase is a Rephelicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at position187 of the modified helicase amino acid sequence, and the helicase is aPcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is present in anamino acid sequence having at least 20% amino acid sequence identity toSEQ ID NO:13 or SEQ ID NO:14, and the helicase is a Rep helicase, orhomolog thereof.

In certain exemplary embodiments, the second amino acid is present in anamino acid sequence having at least 20% amino acid sequence identity toSEQ ID NO:15 or SEQ ID NO:16, and the helicase is a Rep helicase, orhomolog thereof.

In certain exemplary embodiments, the second amino acid residue is atany one of positions 388-411, 422-444 and 518-540 of the modifiedhelicase amino acid sequence, and the helicase is a Rep, PcrA or UvrDhelicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 397-411, 431-444 or 526-540 of the modified helicase aminoacid sequence, and the helicase is a PcrA helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 388-402, 422-435 or 519-531 of the modified helicase aminoacid sequence, and the helicase is a Rep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 393-407, 427-440 or 523-540 of the modified helicase aminoacid sequence, and the helicase is a UvrD helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at position400 of the modified helicase amino acid sequence, and the helicase is aRep helicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at position409 of the modified helicase amino acid sequence, and the helicase is aPcrA helicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 60-82 of the modified helicase amino acid sequence, and thehelicase is a Rep helicase, or homolog thereof. In certain exemplaryembodiments, the first amino acid is at any one of positions 68-79 ofthe modified helicase amino acid sequence, and the helicase is a Rephelicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 69-89 of the modified helicase amino acid sequence, and thehelicase is a PcrA helicase, or homolog thereof. In certain exemplaryembodiments, the first amino acid is at any one of positions 77-87 ofthe modified helicase amino acid sequence, and the helicase is a PcrAhelicase, or homolog thereof.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 67-87 of the modified helicase amino acid sequence, and thehelicase is a UvrD helicase, or homolog thereof. In certain exemplaryembodiments, the first amino acid is at any one of positions 75-85 ofthe modified helicase amino acid sequence, and the helicase is a UvrDhelicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 509-536 of the modified helicase amino acid sequence, and thehelicase is a Rep helicase, or homolog thereof. In certain exemplaryembodiments, the second amino acid is at any one of positions 519-525 ofthe modified helicase amino acid sequence, and the helicase is a Rephelicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 516-534 of the modified helicase amino acid sequence, and thehelicase is a PcrA helicase, or homolog thereof. In certain exemplaryembodiments, the second amino acid is at any one of positions 526-532 ofthe modified helicase amino acid sequence, and the helicase is a PcrAhelicase, or homolog thereof.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 513-531 of the modified helicase amino acid sequence, and thehelicase is a UvrD helicase, or homolog thereof. In certain exemplaryembodiments, the second amino acid is at any one of positions 523-529 ofthe modified helicase amino acid sequence, and the helicase is a UvrDhelicase, or homolog thereof.

In certain exemplary embodiments, said first subdomain and said secondsubdomain comprise no more than a total of two cysteine residues.

In certain exemplary embodiments, the helicase comprises one cysteineresidue and/or is from a bacterium selected from the group consisting ofDeinococcus geothermalis, Meiothermus sp., Marinithermus hydrothermalis,Marinithermus hydrothermalis and Oceanithermus profundus.

In certain exemplary embodiments, the helicase comprises one cysteineresidue or no cysteine residues and/or is from a bacterium selected fromthe group consisting of Thermococcus sp. EXT9, Thermococcus sp. IRI48Thermococcus sp. IRI33, Thermococcus sp. AMT7, Thermococcus nautili,Thermococcus onnurineus (strain NA1), Thermococcus kodakarensis (strainATCC BAA-918/JCM 12380/KOD1) (Pyrococcus kodakaraensis (strain KOD1)),Thermococcus sibiricus (strain MM 739/DSM 12597), Thermococcusparalvinellae, Thermus aquaticus Y51MC23, Thermus aquaticus Y51MC23,Thermus aquaticus Y51MC23, Thermus sp. RL, Thermus sp. RL, Thermus sp.2.9, Salinisphaera hydrothermalis C41B8, Thermus filiformis, Meiothermusruber, Thermus sp. NMX2.A1, Thermus thermophilus JL-18, Thermusscotoductus (strain ATCC 700910/SA-01), Thermus scotoductus (strain ATCC700910/SA-01), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKMB-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKMB-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKMB-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKMB-2274/506), Oceanithermus profundus (strain DSM 14977/NBRC 100410/VKMB-2274506), Thermus oshimai JL-2, Thermus oshimai JL-2, Thermus oshimaiJL-2, Thermomonospora curvata (strain ATCC 19995/DSM 43183/JCM3096/NCIMB 10081), Thermodesulfatator indicus (strain DSM 15286/JCM11887/CIR29812), Geobacillus stearothermophilus (Bacillusstearothermophilus), Coprothermobacter proteolyticus (strain ATCC35245/DSM 5265/BT), Meiothermus silvanus (strain ATCC 700542/DSM9946/VI-R2) (Thermus silvanus), Anaerolinea thermophila (strain DSM14523/JCM 11388/NBRC 100420/UNI-1), Thermoanaerobacteriumthermosaccharolyticum M0795, Meiothermus ruber (strain ATCC 35948/DSM1279/VKM B-1258/21) (Thermus ruber), Meiothermus ruber (strain ATCC35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Deinococcus radiodurans(strain ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB9279/R1/VKM B-1422), Thermodesulfobium narugense DSM 14796, Thermusthermophilus (strain HB8/ATCC 27634/DSM 579), Dictyoglomus thermophilum(strain ATCC 35947/DSM 3960/H-6-12), Thermus thermophilus (strainSG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermusthermophilus (strain SG0.5JP17-16), Thermus sp. CCB_US3_UF1, Deinococcusgeothermalis (strain DSM 11300), Thermus thermophilus (strain HB27/ATCCBAA-163/DSM 7039), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM7039), Marinithermus hydrothermalis (strain DSM 14884/JCM 11576/T1).

In certain exemplary embodiments, the first amino acid and the secondamino acid are each independently an unnatural amino acid or a naturalamino acid.

In certain exemplary embodiments, one or more of an amino acid of thehelicase is substituted with an unnatural amino acid or a natural aminoacid (e.g., a cysteine or a homocysteine).

In certain exemplary embodiments, said helicase comprises a sequenceselected from SEQ ID NOs:4 and 12.

In certain exemplary embodiments, the first amino acid is covalentlycrosslinked to the second amino acid by a disulfide bond or by achemical crosslinker (e.g., a chemical crosslinker having a length offrom about 6 Å to about 25 Å).

In certain exemplary embodiments, the chemical crosslinker is ahis-maleimide crosslinker.

In certain exemplary embodiments, the chemical crosslinker is selectedfrom the group consisting of

1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione,

1-[6-(2,5-dioxopyrrol-1-yl)hexyl]pyrrole-2,5-dione,

1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)phenyl]pyrrole-2,5-dione, and

N,N′-bis[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyl]-N,N′-diphenylbutanediamide.

In certain exemplary embodiments, the chemical crosslinker is

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione.

In one aspect, a modified helicase comprising a first subdomain having afirst amino acid and a second subdomain having a second amino acid,wherein said first amino acid is at least about 30 Å from said secondamino acid when the helicase is in an inactive conformation, and saidfirst amino acid is less than about 20 Å from said second amino acidwhen the helicase is in an active conformation, and wherein a side chainof the first amino acid is chemically crosslinked to a side chain of thesecond amino acid using

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione to form an active,conformationally-constrained helicase is provided.

In another aspect, a modified Rep, PcrA or UvrD helicase or homologthereof, comprising a first subdomain having a first amino acid at anyone of positions 84-116 and a second subdomain having a second aminoacid at any one of positions 388-411, 422-444 and 518-540, wherein aside chain of the first amino acid is covalently crosslinked to a sidechain of the second amino acid with a linker to form an active,conformationally-constrained Rep, PcrA or UvrD helicase, or homologthereof is provided.

In another aspect, a modified Rep helicase or homolog thereof comprisingan amino acid at position 178 covalently crosslinked to an amino acid atposition 400 to form an active, conformationally-constrained Rephelicase or homolog thereof is provided.

In another aspect, a modified Rep helicase or homolog thereof comprisingan amino acid at position 187 covalently crosslinked to an amino acid atposition 409, to form an active, conformationally-constrained helicaseis provided.

In another aspect, a modified helicase comprising a first subdomainhaving a first amino acid and a second subdomain having a second aminoacid, wherein said first amino acid is at least about 30 Å from saidsecond amino acid when the helicase is in an inactive conformation, andsaid first amino acid is less than about 20 Å from said second aminoacid when the helicase is in an active conformation, and wherein a sidechain of the first amino acid is covalently crosslinked to a side chainof the second amino acid with a chemical crosslinker to form an active,conformationally-constrained helicase, and wherein one or more of anamino acid of the helicase is substituted with an unnatural amino acidor a natural amino acid is provided.

In one aspect, a method of making an active,conformationally-constrained helicase is provided. The method includesthe steps of selecting in a helicase a first amino acid in a firstsubdomain that is at least about 30 Å from a second amino acid in asecond subdomain when the helicase is in an inactive conformation, andthe first amino acid is less than about 20 Å from the second amino acidwhen the helicase is in an active conformation, and covalentlycrosslinking the first amino acid to the second amino acid when thehelicase is in an active conformation to form an active,conformationally-constrained helicase.

In a certain exemplary embodiment, the method includes two steps. Thefirst step includes expressing a helicase polypeptide having thecapability to be constrained in a conformation by an intramolecularcrosslinking agent from an isolated nucleic acid selected from a groupconsisting of SEQ ID NOs: 2, 3, 5 and 6. The second step includesreacting the helicase polypeptide with an intramolecular crosslinkingagent to form the conformationally-constrained helicase.

In certain exemplary embodiments, the modified helicase is a SuperFamily 1 (SF1) helicase (e.g., SF1A or SF1B) or a Super Family 2 (SF2)helicase.

In certain exemplary embodiments, the first subdomain comprises a 1Asubdomain or a 1B subdomain and the second subdomain comprises a 2Bsubdomain.

In certain exemplary embodiments, the first amino acid is less thanabout 20 Å, about 19 Å, about 18 Å, about 17 Å, about 16 Å, about 15 Å,about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 5 Å, or about 4 Åfrom the second amino acid when the helicase is in an activeconformation.

In certain exemplary embodiments, the first amino acid is at least about30 Å, about 35 Å, about 40 Å, about 45 Å, about 50 Å, about 55 Å, about60 Å, about 65 Å, about 70 Å, about 75 Å, about 80 Å or about 85 Å fromthe second amino acid when the helicase is in an inactive conformation.

In certain exemplary embodiments, the helicase is selected from thegroup consisting of a Rep helicase, a UvrD helicase and a PcrA helicase.

In certain exemplary embodiments, the helicase comprises a sequenceselected from SEQ ID NOs:4 and 12.

In certain exemplary embodiments, the first amino acid is covalentlylinked to the second amino acid by a disulfide bond or a chemicalcrosslinker.

In another aspect, a method of catalyzing an unwinding reaction of adouble-stranded DNA, comprising contacting the double-stranded DNA witha modified helicase comprising a first subdomain having a first aminoacid and a second subdomain having a second amino acid is provided. Saidfirst amino acid is at least about 30 Å from said second amino acid whenthe helicase is in an inactive conformation, and said first amino acidis less than about 20 Å from said second amino acid when the helicase isin an active conformation. A side chain of the first amino acid iscovalently crosslinked to a side chain of the second amino acid with alinker to form an active, conformationally-constrained helicase.

In certain exemplary embodiments, the conformationally-constrainedhelicase comprises SEQ ID NO: 4 or SEQ ID NO:12.

In certain exemplary embodiments, the conformationally-constrainedhelicase is chemically crosslinked.

In certain exemplary embodiments, the linker comprises an alkyl having alength in the range from C7 to C23 or from C8 to C13.

In another aspect, a method of performing isothermal DNA amplification,comprising combining a DNA template, the conformationally-constrainedhelicase described above and amplification reagents. under conditionscompatible for performing isothermal DNA amplification.

In certain exemplary embodiments, the method includes two steps. Thefirst step includes forming a mixture. The mixture includes adouble-stranded DNA template having a first strand and a second strand;a conformationally-constrained helicase; a DNA-dependent DNA polymerase;a first oligonucleotide primer complementary to a portion of the firststrand; a second oligonucleotide primer complementary to a portion ofthe second strand; and an amplification buffer cocktail. The second stepincludes incubating the mixture at a temperature compatible foractivating the conformationally-constrained helicase and DNA-dependentDNA polymerase.

In certain exemplary embodiments, the conformationally-constrainedhelicase comprises SEQ ID NO:4 or 12. In certain exemplary embodiments,the DNA-dependent DNA polymerase is selected from a group consisting ofE. coli DNA Pol I, E. coli DNA Pol Large Fragment, Bst 2.0 DNAPolymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, BsuDNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase,PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase.

In certain exemplary embodiments, the conformationally-constrainedhelicase is chemically crosslinked.

In certain exemplary embodiments, the chemical crosslinker comprises alength in the range from about 6 Å to about 25 Å.

In certain exemplary embodiments, the chemical crosslinker comprises analkyl having a length in the range from C7 to C23 or from C8 to C13.

In another aspect, a kit for performing helicase dependent amplificationis provided. The kit includes a conformationally-constrained helicaseand amplification reagents (e.g., an amplification buffer cocktail).

In certain exemplary embodiments, the conformationally-constrainedhelicase is selected from SEQ ID NOs: 4 and 12.

In certain exemplary embodiments, the kit further comprising aDNA-dependent DNA polymerase, e.g., selected from a group consisting ofE. coli DNA Pol I, E. coli DNA Pol I Large Fragment, Bst 2.0 DNAPolymerase, Bst DNA Polymerase, Bst DNA Polymerase Large Fragment, BsuDNA Polymerase I Large Fragment, T4 DNA Polymerase, T7 DNA polymerase,PyroPhage® 3173 DNA Polymerase and phi29 DNA Polymerase.

In one aspect, an isolated nucleic acid encoding a modified helicasedescribed herein is provided.

In certain exemplary embodiments, the isolated nucleic acid is selectedfrom the group consisting of SEQ ID NOs: 2, 3, 10 and 11.

In one aspect, a modified E. coli. Rep helicase comprising a firstsubdomain having a first amino acid, a second subdomain having a secondamino acid, and an axis vector defined by the alpha carbon of ILE371from which the vector originates and the alpha carbon of SER280 or thealpha carbon of ALA603, wherein theta is an angle of rotation of saidfirst amino acid and said second amino acid around the axis vector isprovided. A first theta between said first amino acid and said secondamino acid is between about 60 degrees and about 155 degrees when thehelicase is in an inactive conformation, and a second theta between saidfirst amino acid and said second amino acid is between about 355 degreesand about 25 degrees when the helicase is in an active conformation. Aside chain of the first amino acid is covalently crosslinked to a sidechain of the second amino acid with a linker to form an active,conformationally-constrained helicase.

In certain exemplary embodiments, the first theta is about 133 degreesand/or the second theta is about 0 degrees.

In certain exemplary embodiments, the axis vector is defined by thealpha carbon of ILE371 and the alpha carbon of SER280.

In certain exemplary embodiments, the first amino acid is at any one ofpositions 84-108 or 169-187 or at position 178 of the modified helicaseamino acid sequence. In certain exemplary embodiments, the first aminoacid is present in an amino acid sequence having at least 20% amino acidsequence identity to SEQ ID NO:13 or SEQ ID NO:14. In certain exemplaryembodiments, the first amino acid is at any one of positions 60-82 ofthe modified helicase amino acid sequence. In certain exemplaryembodiments, the first amino acid is at any one of positions 68-79 ofthe modified helicase amino acid sequence.

In certain exemplary embodiments, the second amino acid is at any one ofpositions 388-402, 422-435 or 519-531 or at position 400 of the modifiedhelicase amino acid sequence. In certain exemplary embodiments, thefirst amino acid is present in an amino acid sequence having at least20% amino acid sequence identity to SEQ ID NO:15 or SEQ ID NO:16. Incertain exemplary embodiments, the second amino acid is at any one ofpositions 509-536 of the modified helicase amino acid sequence. Incertain exemplary embodiments, the second amino acid is at any one ofpositions 519-525 of the modified helicase amino acid sequence.

These and other features, objects and advantages of the presentinvention will become better understood from the description thatfollows. In the description, reference is made to the accompanyingdrawings, which form a part hereof and in which there is shown by way ofillustration, not limitation, embodiments of the invention.

BRIEF DESCRIPTION OF TILE DRAWINGS

FIG. 1A depicts the closed form Rep crystal structure (PDB entry 1UAA),wherein subdomains are colored and named accordingly and 3′ end of thessDNA (gray) is visible. Residues that were mutated to cysteine andcrosslinked to lock the conformation are shown as pink, blue and red vander Waals spheres in both conformations as reference. Boxed area ismagnified view showing the two residues that were crosslinked forengineering Rep-X.

FIG. 1B depicts the open form Rep crystal structure (PDB entry 1UAA),wherein subdomains are colored and named accordingly and 3′ end of thessDNA (gray) is visible. Residues that were mutated to cysteine andcrosslinked to lock the conformation are shown as pink, blue and red vander Waals spheres in both conformations as reference. Boxed area ismagnified view showing the two residues that were crosslinked forengineering Rep-Y.

FIG. 1C depicts a schematic showing that helicase-catalyzed unwinding ofa DNA labeled with a donor and an acceptor would convert high FRETefficiency (E_(FRET)) to low E_(FRET). Shading level of the donor andacceptor color represents the relative intensity changes. Figurediscloses “(dT)₁₀” as SEQ ID NO: 33.

FIG. 10 depicts an ensemble unwinding kinetics of DNA from FIG. 1C byRep and Rep-X shows the enhanced helicase activity of Rep-X over Rep asmeasured via ensemble E_(FRET). Solid lines are fitted exponential decaycurves as guides to the eye.

FIG. 1E depicts exemplary data of ensemble unwinding kinetics of theRep-Y, Rep-X and non-crosslinked Rep using an assay containing 10 nMhelicase, 5 nM 50-bp ensemble unwinding DNA with 3′-(dT)₃₀ (SEQ ID NO:17) overhang in buffer D and 1 mM ATP).

FIG. 2A depicts a schematic of unwinding stages of dual labeled DNA by aRep-X monomer. Color lightness of the donor (green) and acceptor (red)on the DNA represents the change in the emission intensities as theunwinding progresses.

FIG. 2B depicts representative single molecule time traces show the DNAbinding, unwinding and dissociation for the acceptor strand for Rep-X,wherein the donor fluorescence signal is in green, acceptor in red andE_(FRET) in blue.

FIG. 2C depicts representative single molecule time traces showing theDNA binding, unwinding and dissociation for the donor strand for Rep-X,wherein the donor fluorescence signal is in green, acceptor in red andE_(FRET) in blue. Unwinding period is denoted by Δt.

FIG. 2D depicts representative single molecule time traces showing theDNA binding and dissociation behavior for the donor strand for Rep,wherein the donor fluorescence signal is in green, acceptor in red andE_(FRET) in blue.

FIG. 2E depicts representative single molecule time traces showing theDNA binding and dissociation behavior for the donor su and for Rep-Y,wherein the donor fluorescence signal is in green, acceptor in red andE_(FRET) in blue.

FIG. 2F depicts a representative distribution of Rep-X unwinding periodΔt.

FIG. 2G depicts fractions of DNA binding events that led to unwinding(i.e. exhibited an E_(FRET) increase phase) in smFRET experiments forRep, Rep-Y and Rep-X. Error bars represent 95% confidence bounds.

FIG. 3A depicts a schematic of the optical tweezers assay depicts aRep-X molecule tethered to the bead surface that just loaded on the freessDNA overhang and started to unwind the 6-kbp DNA=.

FIG. 3B depicts unwinding traces showing the extent of processiveunwinding by Rep-X on the 6-kbp DNA (colored according to conditions ofoverhang length, SSB and force, and offset for clarity). Background iscolor coordinated with the inset to show the two laminar flows.

FIG. 3C depicts an exemplary distribution of Rep-X unwinding velocities(N=38). Mean velocity of unwinding and the standard deviation for eachmolecule were plotted above (colors as in B). Figure discloses “(dT)₁₀,”“(dT)₁₅” and “(dT)₇₅” as SEQ ID NOS 33-35, respectively.

FIG. 3D depicts exemplary data comparing the fraction of the completeDNA binding events for Rep, Rep-Y and Rep-X. Error bars represent the95% confidence bounds.

FIG. 3E depicts unwinding by five representative Rep-X molecules in thefixed trap assay are plotted. Pulling force increases during unwindingas the Rep-X pulls the beads closer. Tether breaks appear as suddenforce drops.

FIG. 3F depicts exemplary data showing the average of normalizedunwinding velocities of 58 Rep-X molecules plotted against the pullingforce that shows the high force tolerance of the engineeredsuper-helicase Rep-X. Error bars represent standard error of the mean.

FIG. 4A illustrates a consensus sequence alignment of TxGx motif for 27organisms within 10 out 11 families, wherein Cys is present at position96. Leuconostocaceae family species have an alanine at this position.Figure discloses SEQ ID NOS 109-142, respectively, in order ofappearance.

FIG. 4B illustrates a consensus sequence alignment of motif III for 27organisms within 10 families, wherein Cys is present at position 247.Leuconostocaceae family species have an alanine at this position. Figurediscloses SEQ ID NOS 143-176, respectively, in order of appearance.

FIG. 5A depicts exemplary ATPase activity of mutant PcrA before (“PcrA”)and after crosslinking (“PcrA-X”). Error bars represent standarddeviation over multiple preparations.

FIG. 5B depicts exemplary data of an ensemble unwinding assay for PcrA-Xand wild type PcrA. Solid lines are fitted exponential decay curves asvisual guides.

FIG. 6A depicts representative single molecule time traces for DNAbinding and unwinding by PcrA-X monomers.

FIG. 6B depicts representative single molecule time traces for DNAbinding and unwinding by t PcrA monomers, which are incapable of DNAunwinding.

FIG. 6C depicts exemplary data of fractions of enzyme-DNA binding eventsthat led to an unwinding phase fix PcrA and PcrA-X in the smFRET assay.Error bars represent the 95% confidence bounds

FIG. 6D depicts exemplary data showing processive unwinding of 6-kbp DNAby four representative PcrA-X molecules in the optical tweezers assay.Figure discloses “(dT)₁₅” and “(dT)₇₅” as SEQ ID NOS 34 and 35,respectively.

FIG. 6E depicts exemplary data for fractions of enzyme-DNA binding thatled to the unwinding of 6-kbp DNA in the optical tweezers assay. Errorbars represent the 95% confidence bounds

FIG. 6F depicts a schematic (in subpanel (i)) of the conformationaleffect of RepD, a stimulatory partner of PcrA, on PcrA as measured in asmFRET assay and E_(FRET) histograms (sub-panel (ii)) showing that thePcrA bound to RepD adduct is biased toward the closed form (highE_(FRET) population) compared to PcrA bound to the bare ori-D DNA.

FIG. 7A shows an exemplary SDS-PAGE analysis of Rep-Yintra-crosslinking, wherein the typical three-band pattern on SDSpolyacrylamide gels is evident. Rep-X intra-crosslinking pattern isshown for comparison, wherein the dominant middle band is slightlyshifted for Rep-X compared with the corresponding band for Rep-Y. Lanedesignated as Rep is non-crosslinked Rep.

FIG. 7B shows an exemplary SDS-PAGE analysis of Rep-Y intra-crosslinkingin comparison to uncrosslinked Rep (“Rep”). Lane denoted as Rep-Y*depicts β-ME reduced Rep-Y (crosslinked with a di-sulfide crosslinkerDTME).

FIG. 7C shows an exemplary size exclusion chromatography (SEC) elutionprofile for Rep (dotted line) and the Rep-Y sample (solid line).

FIG. 7D shows an exemplary SDS-PAGE analysis of Rep-Y fractions, F1-F7,collected from SEC (FIG. 5C) in comparison with Rep-Y.

FIG. 7E depicts exemplary data of ssDNA dependent ATPase levels of Rep-Yand Rep. Error bars represent standard deviation over multiplepreparations.

FIG. 8 depicts a schematic of an isothermal DNA amplification processcalled helicase dependent amplification, wherein in step 1: DNA helicase(104) contacts a double-stranded DNA (101) to unwind the first andsecond single strands (102 and 103) and first and second oligonucleotideprimers (105 and 106) hybridize to the first and second single strands(102 and 103) respectively; in step 2: DNA-dependent DNA polymerases(107) bind to the 3′-termini of the first and second oligonucleotideprimers (105 and 106) to initiate chain elongation of new strands (108and 109); and in step 3: continued DNA polymerization results in DNAamplification and formation of new double-stranded DNA (110 and 111).

FIG. 9A shows target residues in Rep (SEQ ID NO: 32), PcrA (SEQ ID NO:177) and UvrD (SEQ ID NO: 178), for −X form crosslinking, calculatedbased on the criteria and crystal structures in open (inactive) andclosed (active) conformations. One residue is chosen from 1A or 1Bdomain (shaded), and another from 2B (shaded).

FIG. 9B shows 56 representative Rep homologs/orthologs with 90% identityto and 80% overlap, and the corresponding region of domain 1A wherecrosslinking residues can be chosen. FIGS. 9B-9C disclose SEQ ID NOS179-235, respectively, in order of appearance.

FIG. 9C shows 56 representative Rep homologs/orthologs with 90% identityto and 80% overlap, and the corresponding region of domain 1B wherecrosslinking residues can be chosen.

FIG. 9D shows 56 representative Rep homologs/orthologs with 90% identityto and 80% overlap, and the corresponding region of domain 2B wherecrosslinking residues can be chosen. FIGS. 9D-9F disclose SEQ ID NOS236-292, respectively, in order of appearance.

FIG. 9E shows 56 representative Rep homologs/orthologs with 90% identityto and 80% overlap, and the corresponding region of domain 2B wherecrosslinking residues can be chosen in addition to those shown in FIG.9D.

FIG. 9F shows 56 representative Rep homologs/orthologs with 90% identityto and 80% overlap, and the corresponding region of domain 2B wherecrosslinking residues can be chosen in addition to those shown in FIG.9E.

FIG. 9G shows target residues in drUvrD, Rep, PcrA and UvrD, for −X formcrosslinking, calculated based on the criteria and crystal structures inopen (inactive) and closed (active) conformations. One residue is chosenfrom 1A or 1B domain (shaded), and another from 2B (shaded). Figurediscloses SEQ ID NOS 293-304, respectively, in order of appearance.

FIG. 10 shows the reaction of maleimide-activated compounds tosulfhydryl-bearing compounds.

FIG. 11 shows a closed form crystal structure of D. radiodurans UvrD(drUvrD; Q9RTI9) with target crosslinking regions of domains 1A, 1B and2B indicated by arrows.

FIG. 12 shows selected target residue pairs for crosslinking, and thespecific distances between the pairs, in a ribbon diagram of a structureof RecD2.

FIG. 13 is a ribbon diagram of a CsRecQ/DNA crystal structure.

FIG. 14 shows a schematic diagram of RecQ DNA helicase, and an overlayof RecQ structures which highlight the mobility of the WH domain.

FIG. 15 shows alternate ribbon diagrams of a RecQ1 crystal structure.

FIG. 16 shows a stereo view of a ribbon diagram of a 5′-3′ SF1superhelicase (T4 Dda).

FIG. 17 shows Rep helicase's 2B domain structure in two differentorientations that differ through a rotation around an axis coming out ofthe plane of the paper. 2B domain orientation can be described by therotation angle θ with respect to the closed form. θ=0 when the helicaseis in the closed form, and θ is 133 degrees when the 2B rotates to theopen form.

DETAILED DESCRIPTION

The present disclosure provides details of the discovery of robustenzymes of the superfamily 1 helicases. The helicase enzymes areengineered as crosslinked, conformationally-constrained monomericconfigurations providing enhanced unwinding activity on dsDNAsubstrates. The “super” helicases display inherently strong physicalproperties having superior characteristics to all presently knownnatural helicases. The disclosed helicases have utility in isothermalPCR and helicase-dependent amplification processes, as well as in nextgeneration sequencing applications, including nanopore sequencingmethods and the like.

Terminology and Definitions

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. With respect tothe use of plural and/or singular toms herein, those having skill in theart can translate from the plural as is appropriate to the contextand/or application. The various singular/plural permutations may beexpressly set forth herein for the sake of clarity.

Terms used herein are intended as “open” terms (e.g., the term“including” should be interpreted as “including but not limited to,” theterm “having” should be interpreted as “having at least,” the term“includes” should be interpreted as “includes but is not limited to,”etc.).

Furthermore, in those instances where a convention analogous to “atleast one of A, B and C, etc.” is used, in general such a constructionis intended in the sense of one having ordinary skill in the art wouldunderstand the convention (e.g., “a system having at least one of A, Band C” would include but not be limited to systems that have A alone, Balone, C alone, A and B together, A and C together, B and C together,and/or A, B, and C together). It will be further understood by thosewithin the art that virtually any disjunctive word and/or phrasepresenting two or more alternative terms, whether in the description orfigures, should be understood to contemplate the possibilities ofincluding one of the terms, either of the terms, or both terms. Forexample, the phrase “A or B” will be understood to include thepossibilities of “A” or ‘B or “A and B.”

All language such as “up to,” “at least,” “greater than,” “less than,”and the like, include the number recited and refer to ranges which cansubsequently be broken down into sub-ranges.

A range includes each individual member. Thus, for example, a grouphaving 1-3 members refers to groups having 1, 2, or 3 members.Similarly, a group having 1-6 members refers to groups having 1, 2, 3,4, or 6 members, and so forth.

The modal verb “may” refers to the preferred use or selection of one ormore options or choices among the several described embodiments orfeatures contained within the same. Where no options or choices aredisclosed regarding a particular embodiment or feature contained in thesame, the modal verb “may” refers to an affirmative act regarding how tomake or use and aspect of a described embodiment or feature contained inthe same, or a definitive decision to use a specific skill regarding adescribed embodiment or feature contained in the same. In this lattercontext, the modal verb “may” has the same meaning and connotation asthe auxiliary verb “can.”

The present invention provides modified helicases that have enhancedenzymatic activity. As used herein, a “helicase” refers to a class ofenzymes that function as motor proteins which move directionally along anucleic acid phosphodiester backbone, separating two annealed nucleicacid strands (i.e., DNA, RNA, or RNA-DNA hybrid) using energy derivedfrom ATP hydrolysis. Helicases are often used to separate strands of aDNA double helix or a self-annealed RNA molecule using the energy fromATP hydrolysis, a process characterized by the breaking of hydrogenbonds between annealed nucleotide bases. They also function to removenucleic acid-associated proteins and catalyze homologous DNArecombination. Metabolic processes of RNA such as translation,transcription, ribosome biogenesis, RNA splicing, RNA transport, RNAediting, and RNA degradation are all facilitated by helicases. Helicasesmove incrementally along one nucleic acid strand of the duplex with adirectionality and processivity specific to each particular enzyme.

Six super families of helicases are known in the art that are classifiedbased on their shared sequence motifs. Helicases not forming a ringstructure are classified in Super Families 1 (SF1) and 2 (SF2).Ring-forming helicases form Super Families 3 (SF3), 4 (SF4), 5 (SF5) and6 (SF6).

SF1 is further subdivided into SF1A and SF1B helicases. In this group,helicases can have either 3′-5′ (SF1A subfamily) or 5′-3′(SF1Bsubfamily) translocation polarity. SF1A helicases include, but are notlimited to are Rep and UvrD in gram-negative bacteria and PcrA helicasefrom gram-positive bacteria. SF1B helicases include, but are not limitedto RecD and Dda helicases.

SF2 is the largest family of helicases, which are involved in variedcellular processes. They are characterized by the presence of nineconserved motifs: Q, I, Ia, Ib, and II through VI. This family primarilycomprises DEAD-box RNA helicases (“DEAD” disclosed as SEQ ID NO: 18).Other helicases in SF2 family are the RecQ-like family and Snf2-likeenzymes. Most of the SF2 helicases are type A, with a few exceptionssuch as the XPD family.

SF3 comprises helicases encoded mainly by small DNA viruses and somelarge nucleocytoplasmic DNA viruses. They have a 3′-5′ translocationdirectionality (therefore they are all type A helicases). SF3 helicaseinclude viral helicases such as the papilloma virus E1 helicase.

SF4 helicases have a type B polarity (5′-3′), and function in bacterialor bacteriophage DNA replication. Gp4 from bacteriophage T7 is an SF4helicase.

SF5 helicases have a type B polarity (5′-3′), and include only thebacterial termination factors Rho.

SF6 helicases contain the core AAA+ that is not included in the SF3classification. SF6 helicases include, but are not limited to, MiniChromosome Maintenance (MCM), RuvB, RuvA, and RuvC.

Exemplary helicases according to the invention include, but are notlimited to RecD, Upf1, PcrA, Rep, UvrD, Hel308, Mtr4, XPD, NS3, Mssl 16,Prp43, RecG, RecQ, TIR, RapA, Hef, RecB, Pif1, Dna2, Dda, Ul5, RecD2,Tral, Sen1p, SETX, IBP160, ZNFX1, Upf1p, UPF1, Hcs1p, IGHMBP2, Dna2p,DNA2, Mtt1p, MOV10, MOV10L1, HELZ, PR285, ptMRDFL1 and the like.

In certain embodiments of the invention, a helicase comprisessubdomains. For example, SF1 helicases comprise subdomains 1A, 1B, 2Aand 2B. The 2B subdomain has been shown to rotate between an openconformation and a closed conformation.

As used herein, an “open conformation” refers to the inactiveconformation of a helicase in which minimal or no helicase activityoccurs. As used herein, a “closed conformation” refers to the activeform of a helicase which has helicase activity. Crystal structuresdepicting the open and/or closed conformations of many helicases havebeen published in the art.

As described further herein, it has been discovered that, by stabilizingthe active (i.e., closed) conformation and destabilizing the inactive(i.e., open) conformation, a modified helicase can be obtained havinggreatly enhanced helicase activity and strength relative to thecorresponding unmodified helicase. According to certain embodiments ofthe invention, a modified helicase that stabilizes the active (i.e.,closed) conformation and destabilizes the inactive (i.e., open)conformation can be generated by covalently linking one or more aminoacids in the 2B subdomain to one or more amino acids in the 1A and/orthe 1B domain of the helicase. Such a modified helicase is referred toherein as an “active, conformationally constrained helicase” or a“helicase-_(X) polypeptide.” Exemplary helicase-_(X) polypeptidesinclude, but are not limited to, Rep-_(X), PcrA-_(X) and UvrD-_(X). Incertain embodiments, a helicase-_(X) polypeptide forms a loop around atarget nucleic acid sequence (e.g., a DNA sequence) In otherembodiments, a helicase-_(X) polypeptide does not form a loop around atarget nucleic acid sequence (e.g., a DNA sequence).

In other embodiments, a helicase is provided that is stabilized in itsinactive (i.e., open) conformation and destabilized in its active (i.e.,closed) conformation. Such a helicase is referred to as an “inactive,conformationally constrained helicase” or a “helicase-_(Y) polypeptide.”Helicase-_(Y) polypeptides exhibit little or no helicase activity.

In certain embodiments, a helicase-_(X) polypeptide has an increasednucleic acid (e.g., DNA) unwinding activity relative to a correspondingunmodified helicase. In certain aspects, the number of base pairs thatcan be unwound by a helicase-_(X) polypeptide is increased by about1000%, about 10,000%, about 100,000% or more (or any ranges or pointswithin the ranges) relative to a corresponding unmodified helicase.

In certain embodiments, a helicase-_(X) polypeptide can unwind at leastabout 500 base pairs, about 1000 base pairs, about 1500 base pairs,about 2000 base pairs, about 2500 base pairs, about 3000 base pairs,about 3500 base pairs, about 4000 base pairs, about 4500 base pairs,about 5000 base pairs, about 5500 base pairs, about 6000 base pairs,about 6500 base pairs, about 7000 base pairs, about 7500 base pairs,about 8000 base pairs, about 8500 base pairs, about 9000 base pairs,about 9500 base pairs, about 10,000 base pairs or more (or any ranges orpoints within the ranges) without dissociating from the nucleic acidsequence (e.g., DNA).

In certain embodiments, a helicase-_(X) polypeptide is stronger that thecorresponding unmodified helicase, withstanding opposing forces of atleast about 10 pN, about 15 pN, about 20 pN, about 25 pN, about 30 pN,about 35 pN, about 40 pN, about 45 pN, about 50 pN, about 55 pN, about60 pN, or more (or any ranges or points within the ranges).

In certain embodiments, a helicase-_(X) polypeptide comprises a firstsubdomain comprising a first amino acid and a second subdomaincomprising a second amino acid, wherein the first amino acid is at leastabout 35 Å from the second amino acid when the helicase is in aninactive conformation, and wherein the first amino acid is less thanabout 25 Å from the second amino acid when the helicase is in an activeconformation. In certain embodiments, the first amino acid is at leastabout 40 Å, about 45 Å, about 50 Å, about 55 Å, about 60 Å, about 65 Å,about 70 Å, about 75 Å, about 80 Å, about 85 Å, or more from the secondamino acid (or any ranges or points within these ranges) when thehelicase is in an inactive (i.e., open) conformation. In certainembodiments, the first amino acid is at most about 20 Å, about 15 Å,about 10 Å, about 9 Å, about 8 Å, about 7 Å, about 6 Å, about 5 Å, about4 Å, or less from the second amino acid (or any ranges or points withinthe ranges) when the helicase is in an active (i.e., closed)conformation. In certain embodiments, the linker in a helicase_(X)polypeptide has a length in the range from about 6 Å to about 25 Å.

In certain embodiments, the first amino acid of a helicase-_(X)polypeptide is present in a 1A or a 1B subdomain and the second aminoacid of a helicase_(X) polypeptide is present in a 2B subdomain.

In certain embodiments, the Rep-_(X) polypeptide forms a loop around thetarget nucleic acid (e.g., DNA) sequence. In certain embodiments, thefirst amino acid of a Rep-_(X) polypeptide that forms a loop is at anyone of positions 84-108 or 169-187, or at position 178 of the Rep aminoacid sequence. In certain embodiments, the second amino acid of aRep_(X) polypeptide that forms a loop is at any one of positions388-402, 422-435 or 519-536, or at position 400 of the Rep amino acidsequence.

In certain embodiments, the PcrA-_(X) polypeptide forms a loop aroundthe target nucleic acid (e.g., DNA) sequence. In certain embodiments,the first amino acid of a PcrA-_(X) polypeptide that forms a loop is atany one of positions 92-116 or 178-196, or at position 187 of the PcrAamino acid sequence. In certain embodiments, the second amino acid of aPcrA-_(X) polypeptide that forms a loop is at any one of positions397-411, 431-444 or 526-540, or at position 409 of the PcrA amino acidsequence.

In certain embodiments, the UvrD-_(X) polypeptide forms a loop aroundthe target nucleic acid (e.g., DNA) sequence. In certain embodiments,the first amino acid of a UvrD-_(X) polypeptide that forms a loop is atany one of positions 90-114 or 175-193 of the UvrD amino acid sequence.In certain embodiments, the second amino acid of a UvrD-_(X) polypeptidethat forms a loop is at any one of positions 393-407, 427-440 or 523-540of the UvrD amino acid sequence.

In certain embodiments, the Rep-_(X) polypeptide does not form a looparound the target nucleic acid (e.g., DNA) sequence. In certainembodiments, the first amino acid of the Rep-_(X) polypeptide that doesnot form a loop is at any one of positions 60-82 (i.e., at any one ofAREMKERVGQTLGRKEARGLMIS (SEQ ID NO: 19)), or at any one of positions68-79 (i.e., at any one of GQTLGRKEARGL (SEQ ID NO: 20)) of the Repamino acid sequence. In certain embodiments, the second amino acid ofthe Rep-_(X) polypeptide that does not form a loop is at any one ofpositions 509-536 (i.e., at any one of FSWMTEMLEGSELDEPMTLTQVVTRFTL (SEQID NO: 21)), or at any one of positions 519-525 (i.e., at any one ofSELDEPM (SEQ ID NO: 22)) of the Rep amino acid sequence.

In certain embodiments, the PcrA-_(X) polypeptide does not form a looparound the target nucleic acid (e.g., DNA) sequence. In certainembodiments, the first amino acid of the PcrA-_(X) polypeptide that doesnot form a loop is at any one of positions 69-89 (i.e., at any one ofAREMRERVQSLLGGAAEDVWI (SEQ ID NO: 23)), or at any one of positions 77-87(i.e., at any one of QSLLGGAAEDV (SEQ ID NO: 24)) of the PcrA amino acidsequence. In certain embodiments, the second amino acid of the PcrA-_(X)polypeptide that does not form a loop is at any one of positions 516-534(i.e., at any one of LSVTKHFENVSDDKSLIAF (SEQ ID NO: 25)), or at any oneof positions 526-532 (i.e., at any one of SDDKSLI (SEQ ID NO: 26)) ofthe PcrA amino acid sequence.

In certain embodiments, the UvrD-_(X) polypeptide does not form a looparound the target nucleic acid (e.g., DNA) sequence. In certainembodiments, the first amino acid of the UvrD-_(X) polypeptide that doesnot form a loop is at any one of positions 67-87 (i.e., at any one ofAAEMRHRIGQLMGTSQGGMWV (SEQ ID NO: 27)), or at any one of positions 75-85(i.e., at any one of GQLMGTSQGGM (SEQ ID NO: 28)) of the UvrD amino acidsequence. In certain embodiments, the second amino acid of the UvrD-_(X)polypeptide that does not form a loop is at any one of positions 513-531(i.e., at any one of VTATRQFSYNEEDEDLMPL (SEQ ID NO: 29)), or at any oneof positions 523-529 (i.e., at any one of EEDEDLM (SEQ ID NO: 30)) ofthe UvrD amino acid sequence.

In certain embodiments, the first amino acid and/or the second aminoacid of a helicase-_(X) polypeptide is present in a particular aminoacid sequence having at least about 15%, about 20%, about 25%, about30%, about 35%, about 40%, about 45 about 50%, about 55%, about 60%,about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98% or about 99% or more sequence identity to that of a referencesequence (e.g, a Rep helicase, A PcrA helicase, a UvrD helicase, or ahomolog or ortholog thereof).

In certain embodiments, the first amino acid is present in a Rephelicase at an amino acid sequence having at least about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% or more amino acid sequenceidentity (or any ranges or points within the ranges) toFHTLGLDIIKREYAALGMKANFSLF (SEQ ID NO:13). In certain embodiments, thefirst amino acid is present in a Rep helicase at an amino acid sequencehaving at least about 15%, about 20%, about 25%, about 30%, about 35%,about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% or more amino acid sequence identity (or any ranges or pointswithin the ranges) to GLYDAHLKACNVLDFDDLI (SEQ ID NO:14).

In certain embodiments, the second amino acid is present in a Rephelicase at an amino acid sequence having at least about 15%, about 20%,about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99% amino acid sequence identity (orany ranges or points within the ranges) to AYLRVLTNPDDDSAF (SEQ IDNO:15). In certain embodiments, the second amino acid is present in aRep helicase at an amino acid sequence having at least about 15%, about20%, about 25%, about 30%, about 35%, about 40% about 45%, about 50%about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about85%, about 90%, about 91%, about 92% about 93% about 94%, about 95%,about 96%, about 97%, about 98% or about 99% amino acid sequenceidentity (or any ranges or points within the ranges) to GEWAMTRNKSMFTA(SEQ ID NO:16).

Suitable amino acid positions for modifying to engineer helicase-_(X)polypeptides (and homologs and orthologs thereof) according to theinvention can be identified by one of ordinary skill in the art usingthis disclosure and well-known local sequence alignment tools.

Techniques fbr determining nucleic acid and amino acid “sequenceidentity” are known in the art. Typically, such techniques includedetermining the nucleotide sequence of genomic DNA, mRNA or cDNA madefrom an mRNA for a gene and/or determining the amino acid sequence thatit encodes, and comparing one or both of these sequences to a secondnucleotide or amino acid sequence, as appropriate. In general,“identity” refers to an exact nucleotide-to-nucleotide or aminoacid-to-amino acid correspondence of two polynucleotides or polypeptidesequences, respectively. Two or more sequences (polynucleotide or aminoacid) can be compared by determining their “percent identity.” Thepercent identity of two sequences, whether nucleic acid or amino acidsequences, is the number of exact matches between two aligned sequencesdivided by the length of the shorter sequences and multiplied by 100.

An approximate alignment for nucleic acid sequences is provided by thelocal homology algorithm of Smith and Waterman, Advances in AppliedMathematics 2:482-489 (1981). This algorithm can be applied to aminoacid sequences by using the scoring matrix developed by Dayhoff, Atlasof Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.3:353-358, National Biomedical Research Foundation, Washington, D.C.,USA, and normalized by Gribskov (1986) Nucl. Acids Res. 14:6745. Anexemplary implementation of this algorithm to determine percent identityof a sequence is provided by the Genetics Computer Group (Madison, Wis.)in the “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.).

One method of establishing percent identity in the context of thepresent invention is to use the MPSRCH package of programs copyrightedby the University of Edinburgh, developed by John F. Collins and ShaneS. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View,Calif.). From this suite of packages, the Smith-Waterman algorithm canbe employed where default parameters are used for the scoring table (forexample, gap open penalty of 12, gap extension penalty of one, and a gapof six). From the data generated the “match” value reflects “sequenceidentity.” Other suitable programs fbr calculating the percent identityor similarity between sequences are generally known in the art, forexample, another alignment program is BLAST, used with defaultparameters. For example, BLASTN and BLASTP can be used using thefollowing default parameters: genetic code=standard; filter=none;strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50sequences; sort by .dbd.HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swissprotein+Spupdate+PIR. Details of these programs can be found at theNCBI/NLM web site.

In certain embodiments of the invention, a helicase is provided that isconformationally-constrained. The term “conformationally-constrained”refers to a conformation having a least one degree of freedom (that is,motion or range of motion) that is less than a reference conformation.In certain embodiments, a conformationally-constrained helicase has aleast one degree of freedom that is less than a helicase that is notconformationally constrained.

In certain embodiments of the invention, a helicase is constrained via acovalent linkage between two or more amino acids of the helicase. Acovalent linkage is a chemical linkage between two atoms or radicalsformed by the sharing of a pair of electrons (i.e., a single bond), twopairs of electrons (i.e., a double bond) or three pairs of electrons(i.e., a triple bond). Covalent linkages are also known in the art aselection pair interactions or electron pair bonds.

In certain embodiments, a covalent linkage is formed via a crosslinkbetween the side chains of two (or more) amino acids of a polypeptide(e.g., between two (or more) amino acids of a modified helicase).

As used herein the term “crosslink” refers to the joining of two or moremolecules by a covalent bond. Crosslinking can occur via disulfidebonds, e.g., between cysteine residues. Crosslinking can occur via theuse of crosslinking reagents (or chemical crosslinkers), which aremolecules that contain two or more reactive ends capable of chemicallyattaching to specific functional groups (primary amines, sulfhydryls,etc.) on proteins or other molecules.

The terms “intramolecular crosslinking agent” and “chemical crosslinkingagent” refer to a compound that can form covalent bonds via specificfunctional groups (e.g., primary amines, sulfhvdryls, etc.) on proteinsor other molecules. An example of an intramolecular or chemicalcrosslinking agent includes a compound having two bifunctional groups inits structure.

Chemical crosslinkers are known in the art, and are commerciallyavailable (e.g., from Thermo Fisher Scientific, Waltham, Mass.). Incertain embodiments, a crosslinker is cleavable (e.g., by reducing oneor more of the functional groups of the crosslinker). In otherembodiments, a crosslinker is not cleavable.

Examples of chemical crosslinkers include, but are not limited to, thosehaving the following functional groups: maleimide, active esters,succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO),difluoro cycloalkynes and linear alkynes), phosphine (such as those usedin traceless and non-traceless Staudinger ligations), haloacetyl (suchas iodoacetamide), phosgene type reagents, sulfonyl chloride reagents,isothiocyanates, acyl halides, hydrazines, disulphides, vinyl sulfones,aziridines and photoreactive reagents (such as aryl azides,diaziridines). Reactions between amino acids and functional goups may bespontaneous, such as cysteine/maleimide, or may require externalreagents, such as Cu(I) for linking azide and linear alkynes.

Linkers can comprise any molecule that stretches across the distancerequired. Linkers can vary in length from one carbon (phosgene-typelinkers) to many Angstroms. In certain embodiments, the linker includesan alkyl having a length in the range from C₇ to C₂₃. In someembodiments, the linker includes an alkyl having a length in the rangefrom C₈ to C₁₃.

Examples of linear molecules include but are not limited to,polyethyleneglycols (PEGs), polypeptides, polysaccharides,deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleicacid (TNA), glycerol nucleic acid (GNA), saturated and unsaturatedhydrocarbons, and polyamides. These linkers may be inert or reactive, inparticular they may be chemically cleavable at a defined position, ormay be themselves modified with a ligand. In certain embodiments, thelinker is resistant to dithiothreitol (DTT).

Examples of crosslinkers include, but are not limited to2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate,2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate,di-maleimide PEG lk, di-maleimide PEG 3.4k, di-maleimide PEG 5k,di-maleimide PEG 10k, bis(maleimido)ethane (BMOE), bis-maleimidohexane(BMH), 1,4-bis-maleimidobutane (BMB), 1,4bis-maleimidyl-2,3-dihydroxybutane (BMDB), BM[PEO]2(1,8-bis-maleimidodiethyleneglycol), BM[PEO]3(1,11-bis-maleimidotriethylene glycol), tris[2-maleimidoethyl]amine(TMFA), dithiobismaleimidoethane (DTME), bis-maleimide PEG3,bis-maleimide PEGU, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2,DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8 kDa, DBCO-PEG-DBCO 4.0 kDa,DBCO-15 atoms-DBCO, DBCO-26 atoms-DBCO, DBCO-35 atoms-DBCO,DBCO-PEG4-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin, DBCO-S-S-PEG11-biotinand (succinimidyl 3-(2-pyridyldithio)propionate (SPDP).

In certain embodiments, a covalent linkage refers to the linkage betweentwo or more amino acids. One or more of the linked amino acids may benaturally occurring or non-naturally occurring. One or more of thelinked amino acids may be chemically modified.

As used herein, a “natural amino acid” refers to the twenty geneticallyencoded alpha-amino acids. See, e.g., Biochemistry by L. Stryer, 3^(rd)ed. 1988, Freeman and Company, New York for structures of the twentynatural amino acids.

As used herein, an “unnatural amino acid,” “modified amino acid” or“chemically modified amino acid” refers to any amino acid, modifiedamino acid, or amino acid analogue other than the twenty geneticallyencoded alpha-amino acids. Unnatural amino acids have side chain groupsthat distinguish them from the natural amino acids, although unnaturalamino acids can be naturally occurring compounds other than the twentyproteinogenic alpha-amino acids. In addition to side chain groups thatdistinguish them from the natural amino acids, unnatural amino acids mayhave an extended backbone such as beta-amino acids.

Non-limiting examples of unnatural amino acids include selenocysteine,pyrrolysine, homocysteine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, an unnatural analogue of a tyrosine aminoacid; an unnatural analogue of a glutamine amino acid; an unnaturalanalogue of a phenylalanine amino acid; an unnatural analogue of aserine amino acid; an unnatural analogue of a threonine amino acid; analkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl,alkenyl, alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid,borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone,imine, aldehyde, hydroxylamine, keto, or amino substituted amino acid,or any combination thereof; an amino acid with a photoactivatablecross-linker; a spin-labeled amino acid; a fluorescent amino acid; anamino acid with a novel functional group; an amino acid that covalentlyor noncovalently interacts with another molecule; a metal binding aminoacid; a metal-containing amino acid; a radioactive amino acid; aphotocaged and/or photoisomerizable amino acid; a biotin orbiotin-analogue containing amino acid; a glycosylated or carbohydratemodified amino acid; a keto containing amino acid; amino acidscomprising polyethylene glycol or polyether; a heavy atom substitutedamino acid; a chemically cleavable or photocleavable amino acid; anamino acid with an elongated side chain; an amino acid containing atoxic group; a sugar substituted amino acid, e.g., a sugar substitutedserine or the like; a carbon-linked sugar-containing amino acid; aredox-active amino acid; an α-hydroxy containing acid; an amino thioacid containing amino acid; an α,α disubstituted amino acid; a β-aminoacid; and a cyclic amino acid other than proline. In an embodiment ofthe helicases described herein, one or more amino acids of the helicaseare substituted with one or more unnatural amino acids and/or one ormore natural amino acids.

In certain embodiments, a helicase-_(X) is a closed form,conformationally-constrained helicase monomer generated from a helicasepolypeptide that was reacted with an intramolecular crosslinking agent.In certain embodiments, a helicase-_(Y) is an open form,conformationally-constrained helicase monomer generated from a helicasepolypeptide that was reacted with an intramolecular crosslinking agent.

The chemical structures described herein are named according to IUPACnomenclature rules and include art-accepted common names andabbreviations where appropriate. The IUPAC nomenclature can be derivedwith chemical structure drawing software programs, such as ChemDraw®(PerkinElmer, Inc.), ChemDoodle® (iChemLabs, LLC) and Marvin (ChemAxonLtd.). The chemical structure controls in the disclosure to the extentthat an IUPAC name is misnamed or otherwise conflicts with the chemicalstructure disclosed herein. E. coli Rep mutants can be engineered thatare intramolecularly crosslinked to constrain the 2B subdomain in openor closed conformations. Residues for the cysteine substitutionmutagenesis and the length of the bis-maleimide crosslinkers wereselected such that when crosslinked, the 2B subdomain cannot rotateappreciably, effectively locking the protein in one conformation (FIG.1A, B). The closed fix in of a helicase that is crosslinked in aconstrained conformation is denoted with the suffix “-X”, and the openform of a helicase that is crosslinked in a constrained conformation isdenoted with the suffix “-Y.” For Rep, Rep-X and Rep-Y represent theconformationally-constrained closed and open forms, respectively.Enzymatic activities of Rep-X and Rep-Y monomers were studied in singlemolecule and ensemble assays employing fluorescence resonance energytransfer (FRET), total internal reflection fluorescence (TIRF)microscopy, and optical tweezers force spectroscopy.

The Rep mutant sequences used to generate Rep-X and Rep-Y include thosenucleotide and amino acid sequences identified in Table 1.

TABLE 1  Amino Acid and Nucleotide Sequences for exemplary Rep-Xand Rep-Y proteins Polypeptide/DNA/RNA 5′→3′ (nucleotide sequence) (SEQ ID NO:_) N→C (amino acid sequence) Wild type Rep helicaseATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (gene sequence)ACCGGCCCCTGCCTGGTGCTGGCGGGCGCGGGTTCCGGTAAA >gi|556503834:3960677-ACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTT 3962698GCGGTTATCAGGCGCGGCACATTGCGGCGGTGACCTTTACTA Escherichia coliATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACG str. K-12CTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTC substr. MG1655,CATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCG complete genomeCTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATC (SEQ ID NO: 31)AGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAGATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAACTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAGTGCGATTGGCGAGCGGGACCGTATTTTTGCCCATTGTTATGGGCTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCGATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGAAGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCTGGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCTGGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGTGGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACGTCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTGAAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGTATTCTGAAAGCGGCGAACATCCTGATCGCCAATAACCCGCACGTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGGAGCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTGAGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAAAACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCATCAGTCGCGGGTGETTGAAAAATTCCTGATGCAAAACCGCATCCCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTGAAATCAAGCACTTGCTGGCTTATCTGCGCGTGCTGACTAACCCGGACGATGACAGCGCATTTCTGCGTATCGTTAACACGCCGAAGCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTGGGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGATATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGCATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTGGCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCATGGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGCCCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTGTTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGATGAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGCGCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATCAGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGTTTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCCGCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCGGCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATTGACCTTTACGCCTGTGTAAAGAACGCCGTCAGTACGGCGAACTGGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCAGGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGCCGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCTGAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Wild type Rep helicaseMRLNPGQQQAVEFVTGPCLVLAGAGSGKTRVITNKIAHLIRGCG (amino acid sequence)YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLG >gi|48994965|gb LDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQ AAT48209.1|DNA helicase QLISTISNWKNDLKTPSQAAASAIGERDRIFAHCYGLYDAHLKAC and single-stranded NVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLVDEYQDTNTS DNA-dependent ATPaseQYELVKLLVGSRARFTVVGDDDQSIYSWRGARPQNLVLLSQDFP [Escherichia coli str. ALKVIKLEQNYRSSGRILKAANILLANNPHVFEKRLFSELGYGAEL K-12 substr. MG16]KVLSANNEEHEAERVTGELIAHHFVNKTQYKDYAILYRGNHQSR (SEQ ID NO: 32)VFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDSAFLRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLSGRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYETSPSPKAAEMRMKNVNQLFSWMTEMLEGSELDEPMTLTQVVTRFTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEGFLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLCKERRQYGELVRPEPSRFLLELPQDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGKRep-_(X) polypmtide¹ MRLNPGQQQAVEFVTGPLLVLAGAGSGKTRVITNKIAHLIRGSG(SEQ ID NO: 1) YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLGLDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQQLISTISNWKNDLKTPSQAAASAIGERDRIFAHVYGLYDAHLKACNVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLVDEYQDTNTSQYELVKLLVGSRARFTWGDDDQSIYSWRGARPQNLVLLSQDFPALKVIKLEQNYRSSGRILKAANILIANNPHVFEKRLFSELGYGAELKVLSANNEEHEAERVTGELLAHHFVNKTQYKDYAILYRGNHQSRVFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDCAFLRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLSGRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYETSPSPKAAEMRMKNVNQLFSWNTEMLEGSELDEPMTLTQVVTRFTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEGFLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLAKERRQYGELVRPEPSRFLLELPQDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGK Rep-_(X) DNA²ATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (SEQ ID NO: 2)ACCGGCCCCTTGCTGGTGCTGGCGGGCGCGGGTTCCGGTAAAACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTAGCGGGTACCAGGCGCGGCACATTGCGGCGGTGACCTTTACTAATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACGCTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTCCATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCGCTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATCAGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAGATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAACTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAGTGCGATTGGCGAGCGGGACCGTATTTTTGCCCATGTTTATGGGCTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCGATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGAAGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCTGGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCTGGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGTGGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACGTCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTGAAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGTATTCTCAAAGCGGCGAACATCCTGATCGCCAATAACCCGCACGTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGGACTCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTGAGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAAAACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCATCAGTCGCGGGTGTTTGAAAAATTCCTGATGCAAAACCGCATCCCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTGAAATCAAGGACTTGCTGGCTTATCTGCGCGTGCTGACTAACCCGGACGATGACTGCGCATTTCTGCGTATCGTTAACACGCCGAAGCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTGGGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGATATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGCATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTGGCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCATGGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGCCCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTGTTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGATGAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGCGCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATCAGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGTTTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCCGCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCGGCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATTGACCTTTACGCTGGCTAAAGAACGCCGTCAGTACGGCGAACTGGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCAGGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGCCGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCTGAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Rep-_(X) RNA³AUGCGUCUAAACCCCGGCCAACAACAAGCUGUCGANUUCGU (SEQ ID NO: 3)UACCGGCCCCUUGCUGGUGCUGGCGGGCGCGGGUUCCGGUAAAACUCGUGUUAUCACCAAUAAAAUCGCCCAUCUGAUCCGCGGUAGCGGGUACCAGGCGCGGCACAUUGCGGCGGUGACCUUUACUAAUAAAGCAGCGCGCGAGAUGAAAGAGCGUGUAGGGCAGACGCUGGGGCGCAAAGAGGCGCGUGGGCUGAUGAUCUCCACUUUCCAUACGUUGGGGCUGGAUAUCAUCAAACGCGAGUAUGCGGCGCUUGGGAUGAAAGCGAACUUCUCGUUGUUUGACGAUACCGAUCAGCUUGCUUUGCUUAAAGAGUUGACCGAGGGGCUGAUUGAAGAUGACAAAGUUCUCCUGCAACAACUGAUUUCGACCAUCUCUAACUGGAAGAAUGAUCUCAAAACACCGUCCCAGGCGGCAGCAAGUGCGAUUGQCGAGCGGGACCGUAUUUUUGCCCAUGUUUAUGGGCUGUAUGAUGCACACCUGAAAGCCUGUAACGUUCUCGACUUCGAUGAUCUGAUUUUAUUGCCGACGUUGCUGCUGCAACGCAAUGAAGAAGUCCGCAAGCGCUGGCAGAACAAAAUUCGCUAUCUGCUGGUGGAUGAGUAUCAGGACACCAACACCAGCCAGUAUGAGCUGGUGAAACUGCUGGUGGGCAGCCGCGCGCGCUUUACCGUGGUGGGUGACGAUGACCAGUCGAUCUACUCCUGGCGCGGUGCACGUCCGCAAAACCUGGUGCUGCUGAGUCAGGAUUUUCCGGCGCUGAAGGUGAUUAAGCUUGAGCAGAACUAUCGCUCUUCCGUGCGUAUUCUGAAAGCGGCGAACAUCCUGAUCGCCAAUAACCCGCACGUCUUUGAAAAGCGUCUGUUCUCCGAACUGGGUUAUGGCGCGGAGCUAAAAGUAUUAAGCGCGAAUAACGAAGAACAUGAGGCUGAGCGCGUUACUGGCGAGCUGAUCGCCCAUCACUUCGUCAAUAAAACGCAGUACAAAGNUUACGCCAUUCUUUAUCGCGGUAACCAUCAGUCGCGGGUGUUUGAAAAAUUCCUGAUGCAAAACCGCAUCCCGUACAAAAUAUCUGGUGGUACGUCGUUUUUCUCUCGUCCUGAAAUCAAGGACUUGCUGGCUUAUCUGCGCGUGCUGACUAACCCGGACGAUGACUGCGCAUUUCUGCGUAUCGUUAACACGCCGAAGCGAGAGAUUGGCCCGGCUACGCUGAAAAAGCUGGGUGAGUGGGCCAUGACGCGCAAUAAAAGCAUGUUUACCGCCAGCUUUGAUAUGGGCCUGAGUCAGACGCUUAGCGGACGUGGUUAUGAAGCAUUGACCCGCUUCACUCACUGGUUGGCAGAAAUCCAGCGUCUGGCGGAGCGGGAGCCGAUUGCCGCGGUGCGUGAUCUGAUCCAUGGCAUGGAUUAUGAAUCCUGGCUGUACGAAACAUCGCCCAGCCCGAAAGCCGCCGAAAUGCGCAUGAAGAACGUCAACCAACUGUULAGCUGGAUGACGGAGAUGCUGGAAGGCAGUGAACUGGAUGAGCCGAUGACGCUCACCCAGGUGGUGACGCGCUUUACUUUGCGCGACAUGAUGGAGCGUGGUGAGAGUGAAGAAGAGCUGGAUCAGGUGCAACUGAUGACUCUCCACGCGUCGAAAGGGCUGGAGUUUCCUUAUGUCUACAUGGUCGGUAUGGAAGAAGGGUUUUUGCCGCACCAGAGCAGCAUCGAUGAAGAUAAUAUCGAUGAGGAGCGGCGGCUGGCCUAUGUCGGCAUUACCCGCGCCCAGAAGGAAUUGACCUUUACGCUGGCUAAAGAACGCCGUCAGUACGGCGAACUGGUGCGCCCGGAGCCGAGCCGCUUUUUGCUGGAGCUGCCGCAGGAUGAUCUGAUUUGUGAACAGGAGCGCAAAGUGGUCAGCGCCGAAGAACGGAUGCAGAAAGGGCAAAGCCAUCUGGCGAAUCUGAAAGCGAUGAUGGCGGCA AAACGAGGGAAAUAARep-_(X) polypeptide⁴ SEQ ID NO: 1 and formula no 2 in Table 2 (SEQ ID NO: 4) (1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2, 5-dione)Rep-_(Y) polypeptide⁵ MRLNPGQQQAVEFVTGPLLVLAGAGSGKTRVITNKIAHLIRGSG(SEQ ID NO: 5) YQARHIAAVTFTNKAAREMKERVGQTLGRKEARGLMISTFHTLGLDIIKREYAALGMKANFSLFDDTDQLALLKELTEGLIEDDKVLLQQLISTISNWKNDLKTPSQAAASAIGERDRIFAHVYGLYDAHLKACNVLDFDDLILLPTLLLQRNEEVRKRWQNKIRYLLNDEYQDTNTSQYELVKLLVGSRARFTVVGDDDQSIYSWRGARPQNLVLLSQDFPALKVIKLEQNYRSSGRILKAANILIANNPHVFEKRLFSELGYGAELKVLSANNEEHEAERVTGELIAHHFVNKTQYKDYAILYRGNHQSRVFEKFLMQNRIPYKISGGTSFFSRPEIKDLLAYLRVLTNPDDDCAFLRIVNTPKREIGPATLKKLGEWAMTRNKSMFTASFDMGLSQTLSGRGYEALTRFTHWLAEIQRLAEREPIAAVRDLIHGMDYESWLYETSPSPKAAEMRMKNVNQLFSWMTEMLEGSELDEPMTLTQVVTRFTLRDMMERGESEEELDQVQLMTLHASKGLEFPYVYMVGMEEGFLPHQSSIDEDNIDEERRLAYVGITRAQKELTFTLAKERRQYGELVRPEPSRFLLELPDDLIWEQERKVVSAEERMQKGQSHLANLKAM MAAKRGK Rep-_(Y) DNA⁶ATGCGTCTAAACCCCGGCCAACAACAAGCTGTCGAATTCGTT (SEQ ID N0:6)ACCGGCCCCTTGCTGGTGCTGGCGGGCGCGGGTTCCGGTAAAACTCGTGTTATCACCAATAAAATCGCCCATCTGATCCGCGGTAGCGGGTACCAGGCGCGGCACATTGCGGCGGTGACCTTTACTAATAAAGCAGCGCGCGAGATGAAAGAGCGTGTAGGGCAGACGCTGGGGCGCAAAGAGGCGCGTGGGCTGATGATCTCCACTTTCCATACGTTGGGGCTGGATATCATCAAACGCGAGTATGCGGCGCTTGGGATGAAAGCGAACTTCTCGTTGTTTGACGATACCGATCAGCTTGCTTTGCTTAAAGAGTTGACCGAGGGGCTGATTGAAGATGACAAAGTTCTCCTGCAACAACTGATTTCGACCATCTCTAACTGGAAGAATGATCTCAAAACACCGTCCCAGGCGGCAGCAAGTGCGATTGGCGAGCGGGACCGTATTTTTGCCCATGTTTATGGGCTGTATGATGCACACCTGAAAGCCTGTAACGTTCTCGACTTCGATGATCTGATTTTATTGCCGACGTTGCTGCTGCAACGCAATGAAGAAGTCCGCAAGCGCTGGCAGAACAAAATTCGCTATCTGCTGGTGGATGAGTATCAGGACACCAACACCAGCCAGTATGAGCTGGTGAAACTGCTGGTGGGCAGCCGCGCGCGCTTTACCGTGGTGGGTGACGATGACCAGTCGATCTACTCCTGGCGCGGTGCACGTCCGCAAAACCTGGTGCTGCTGAGTCAGGATTTTCCGGCGCTGAAGGTGATTAAGCTTGAGCAGAACTATCGCTCTTCCGGGCGTATTCTGAAAGCGGCGAACATCCTGATCGCCAATAACCCGCACGTCTTTGAAAAGCGTCTGTTCTCCGAACTGGGTTATGGCGCGGAGCTAAAAGTATTAAGCGCGAATAACGAAGAACATGAGGCTGAGCGCGTTACTGGCGAGCTGATCGCCCATCACTTCGTCAATAAAACGCAGTACAAAGATTACGCCATTCTTTATCGCGGTAACCATCAGTCGCGGGTGTTTGAAAAATTCCTGATGCAAAACCGCATCCCGTACAAAATATCTGGTGGTACGTCGTTTTTCTCTCGTCCTGAAATCAAGGACTTGCTGGCTTATCTGCGCGTGCTGACTAACCCGGACGATGACTGCGCATTTCTGCGTATCGTTAACACGCCGAAGCGAGAGATTGGCCCGGCTACGCTGAAAAAGCTGGGTGAGTGGGCGATGACGCGCAATAAAAGCATGTTTACCGCCAGCTTTGATATGGGCCTGAGTCAGACGCTTAGCGGACGTGGTTATGAAGCATTGACCCGCTTCACTCACTGGTTGGCAGAAATCCAGCGTCTGGCGGAGCGGGAGCCGATTGCCGCGGTGCGTGATCTGATCCATGGCATGGATTATGAATCCTGGCTGTACGAAACATCGCCCAGCCCGAAAGCCGCCGAAATGCGCATGAAGAACGTCAACCAACTGTTTAGCTGGATGACGGAGATGCTGGAAGGCAGTGAACTGGATGAGCCGATGACGCTCACCCAGGTGGTGACGCGCTTTACTTTGCGCGACATGATGGAGCGTGGTGAGAGTGAAGAAGAGCTGGATCAGGTGCAACTGATGACTCTCCACGCGTCGAAAGGGCTGGAGTTTCCTTATGTCTACATGGTCGGTATGGAAGAAGGGTTTTTGCCGCACCAGAGCAGCATCGATGAAGATAATATCGATGAGGAGCGGCGGCTGGCCTATGTCGGCATTACCCGCGCCCAGAAGGAATTGACCTTTACGCTGGCTAAAGAACGCCGTCAGTACGGCGAACTGGTGCGCCCGGAGCCGAGCCGCTTTTTGCTGGAGCTGCCGCAGGATGATCTGATTTGGGAACAGGAGCGCAAAGTGGTCAGCGCCGAAGAACGGATGCAGAAAGGGCAAAGCCATCTGGCGAATCTGAAAGCGATGATGGCGGCAAAACGAGGGAAATAA Rep-_(Y) RNA⁷AUGCGUCUAAACCCCGGCCAACAACAAGCUGUCGAAUUCGU (SEQ ID NO: 7)UACCGGCCCCUUGCUGGUGCUGGCGGGCGCGGGUUCCGGUAAAACUCGUGUUAUCACCAAUAAAAUCGCCCAUCUGAUCCGCGGUAGCGGGUACCAGGCGCGGCACAUUGCGGCGGUGACCUUUACUAAUAAAGCAGCGCGCGAGAUGAAAGAGCGUGUAGGGCAGACGCUGGGGCGCAAAGAGGCGCGUGGGCUGAUGAUCUCCACUUUCCAUACGUUGGGGCUGGAUAUCAUCAAACGCGAGUAUGCGGCGCUUGGGAUGAAAGCGAACUUCUCGUUGUUUGACGAUACCGAUCAGCUUGCUUUGCUUAAAGAGUUGACCGAGGGGCUGAUUGAAGAUGACAAAGUUCUCCUGCAACAACUGAUUUCGACCAUCUCUAACUGGAAGAAUGAUCUCAAAACACCGUCCCAGGCGGCAGCAAGUGCGAUUGGCGAGCGGGACCGUAUUUUUGCCCAUGUUUAUGGGCUGUAUGAUGCACACCUGAAAGCCUGUAACGUUCUCGACUUCGAUGAUCUGNUUUUAUUGCCGACGUUGCUGCUGCAACGCAAUGAAGAAGUCCGCAAGCGCUGGCAGAACAAAAUUCGCUAUCUGCUGGUGGAUGAGUAUCAGGACACCAACACCAGCCAGUAUGAGCUGGUGAAACUGCUGGUGGGCAGCCGCGCGCGCUUUACCGUGGUGGGUGACGAUGACCAGUCGAUCUACUCCUGGCGCGGUGCACGUCCGCAAAACCUGGUGCUGCUGAGUCAGGAUUUUCCGGCGCUGAAGGUGAUUAAGCUUGAGCAGAACUAUCGCUCUUCCGGGCGUAUUCUGAAAGCGGCGAACAUCCUGAUCGCCAAUAACCCGCACGUCUUUGAAAAGCGUCUGUUCUCCGAACUGGGUUAUGGCGCGGAGCUAAAAGUAUUAAGCGCGAAUAACGAAGAACAUGAGGCUGAGCGCGUUACUGGCGAGCUGAUCGCCCAUCACUUCGUCAAUAAAACGCAGUACAAAGAUUACGCCAUUCUUUAUCGCGGUAACCAUCAGUCGCGGGUGUUUGAAAAAUUCCUGAUGCAAAACCGCAUCCCGUACAAAAUAUCUGGUGGUACGUCGUUUUUCUCUCGUCCUGAAAUCAAGGACUUGCUGGCUUAUCUGCGCGUGCUGACUAACCCGGACGAUGACUGCGCAUUUCUGCGUAUCGUUAACACGCCGAAGCGAGAGAUUGGCCCGGCUACGCUGAAAAAGCUGGGUGAGUGGGCGAUGACGCGCAAUAAAAGCAUGUUUACCGCCAGCUUUGAUAUGGGCCUGAGUCAGACGCUUAGCGGACGUGGUUAUGAAGCAUUGACCCGCUUCACUCACUGGUUGGCAGAAAUCCAGCGUCUGGCGCAGCGGCAGCCGAUUGCCGCGGUGCGUGAUCUGAUCCAUGGCAUGGAUUAUGAAUCCUGGCUGUACGAAACAUCGCCCAGCCCGAAAGCCGCCGAAAUGCGCAUGAAGAACGUCAACCAACUGUUUAGCUGGAUGACGGAGAUGCUGGAAGGCAGUGAACUGGAUGAGCCGAUGACGCUCACCCAGGUGGUGACGCGCUUUACUUUGCGCGACAUGAUGGAGCGUGGUGAGAGUGAAGAAGAGCUGGAUCAGGUGCAACUGAUGACUCUCCACGCGUCGAAAGGGCUGGAGUUUCCUUAUGUCUACAUGGUCGGUAUGGAAGAAGGGUUUUUGCCGCACCAGAGCAGCAUCGAUGAAGAUAAUAUCCAUGAGGAGCGGCGGCUGGCCUAUGUCGGCAUUACCCGCGCCCAGAAGGAAUUGACCUUUACGCUGGCUAAAGAACGCCGUCAGUACGGCGAACUGGUGCGCCCGGAGCCGAGCCGCUUUUUGCUGGAGCUGCCGCAGGAUGAUCUGAUUUGGGAACAGGAGCGCAAAGUGGUCAGCGCCGAAGAACGGAUGCAGAAAGGGCAAAGCCAUCUGGCGAAUCUGAAAGCGAUGAUGGCGGCA AAACGAGGGAAAUAARep-_(Y) polypeptide⁸ SEQ ID NO: 5 and formula no 2 in Table 2 (SEQ ID NO: 8) (1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2, 5-dione).¹This Rep mutant encodes mutations removing natural cysteine residuesfound in the wild-type Rep and include further amino acid mutations tofacilitate intramolecular crosslinking to an intramolecular crosslinkingagent to generate the Rep-x polypeptide. ²The DNA sequence correspondsto the open reading frame that encodes the polypeptide of SEQ ID NO: 1.³The RNA sequence corresponds to the open reading frame that encodes thepolypeptide of SEQ ID NO: l. ⁴The Rep-_(X) polypeptide closed foimmonomer following reaction of Repx polypeptide (SEQ ID NO: 1) with anintramolecular crosslinking agent: ⁵This Rep mutant encodes mutationsthat remove natural cysteine residues found in the wild-type Rep andinclude further amino acid mutations to facilitate intramolecularcrosslinking to an intramolecular crosslinking agent to generate theRep-y polypeptide. ⁶The DNA sequence corresponds to the open readingframe that encodes the polypeptide of SEQ ID NO: 5. ⁷The RNA sequencecorresponds to the open reading frame that encodes the polypeptide ofSEQ ID NO: 5. ⁸The Rep-_(Y) polypeptide open forui monomer followingreaction of Repy polypeptide (SEQ ID NO: 5) with an intramolecularcrosslinking agent:

The intramolecular crosslinking agents suitable for generating versionsof Rep-_(X) and Rep-_(Y) include those identified in Table 2.

TABLE 2 Exemplary intramolecular crosslinking agents for generatingRep-_(X) and Rep-_(Y) Formula No. Compound Structure (IUPAC Name) 1

2

3

4

5

6

These intramolecular crosslinking agents yield intramolecularcrosslinked monomer structures when reacted with Rep-_(X) and Rep-_(Y)polypeptides. The linkers can have a length in the range from about 6 Åto about 25 Å. These types of linkers have an alkyl length in the rangecorresponding from about C₇ to about C₂₀, wherein highly preferredlinkers have a length in the range from about C₁₀ to about C₁₂. Methodsand conditions for generating intramolecular crosslink formation inproteins are well known in the art for these types of intramolecularcrosslinking agents, and such methods and conditions are applicable tothe helicases of this disclosure.

Rep-_(X) would be inefficient in DNA unwinding even at highconcentrations that make the wild type Rep active if the closed for inis inactive for unwinding. In multiple turnover ensemble unwindingreactions using FRET-labeled DNA (see, for example, FIG. 1C), however,Rep-X unwound an 18-bp substrate with a 3′-(dT)₁₀ overhang (SEQ ID NO:33) at a much faster rate and higher reaction amplitude than the wildtype Rep (FIG. 1D). In contrast, Rep-Y unwinding rates were similar tothat of Rep (FIG. 1E), indicating that the dramatic unwindingenhancement is specifically achieved in the closed conformation. Becausethe large enhancement in unwinding activity observed in bulk solutioncan result from the activation of a monomer or from enhancedoligomerization, single molecule FRET (smFRET) experiments wereperformed to test if a single Rep-X can unwind DNA.

Rep and Rep-X monomers were immobilized to a polymer-passivated quartzsurface using antibodies against the N-terminal hexa-histidine-tag (SEQID NO: 36) on the protein (FIG. 2A) to ensure that the observed activitybelonged to monomers (T. Ha et al., Initiation and re-initiation of DNAunwinding by the Escherichia coli Rep helicase. Nature 419, 638-641(2002)). For the unwinding substrate, we used a 18-bp duplex DNA with a3′-(dT)₂₀ overhang (SEQ ID NO: 37) labeled with a donor (Cy3) and anacceptor (Cy5) at two opposite ends of the DNA duplex, allowing us toidentify unwinding reactions as increases in FRET efficiency (E_(FRET))(FIG. 2A) (G. Lee, M. A. Bratkowski, F. Ding A. Ke, T. Ha, ElasticCoupling Between RNA Degradation and Unwinding by an Exoribonuclease.Science (New York, N.Y. 336, 1726-1729 (2012)). When the DNA and ATPwere added to the reaction chamber, we could observe the capture of asingle DNA molecule by a single protein as the sudden appearance offluorescence signal (FIG. 2B-E). Subsequent DNA unwinding generatedssDNA strands that coil up due to high flexibility and E_(FRET)increased (M. C. Murphy, I. Rasnik, W. Cheng, T. M. Lohman, T. Ha,Probing single-stranded DNA conformational flexibility usingfluorescence spectroscopy. Biophysical journal 86, 2530-2537 (2004)).Once the duplex was completely unwound, the acceptor-labeled strand wasreleased, which was marked by sudden disappearance of the acceptorsignal and recovery of the donor signal. The donor-labeled strand thendissociated, resulting in complete loss of fluorescence. The meanduration of unwinding measured from the E_(FRET) increase to acceptorstrand release was ˜0.6 s, giving a lower limit on the unwinding speedof 30 bp/s for the 18-bp substrate (FIG. 2F). About 82% of the DNAmolecules (661 out of 809) that initially bound to Rep-X monomers wereunwound (FIG. 2G). In contrast, only 2% of the DNA molecules (13 out of847) that bound to Rep (i.e. without crosslinking) showed unwinding, andthe unwinding yield for Rep-Y was 16% (357 out of 2212) (FIG. 2G),showing that constraining Rep into the closed form selectively activatesthe unwinding activity of a monomer. The nonzero amplitude of unwindingfor Rep and Rep-Y may be due to conformational constraints caused bysurface tethering in a small fraction of molecules.

In vitro studies have shown that the unwinding processivity of Rep andrelated helicases is limited even in their oligomeric forms, rangingfrom 30-50 bp (A. Niedziela-Majka, M. A. Chesnik, E. J. Tomko, T. M.Lohman, Bacillus stearothermophilus PcrA monomer is a single-strandedDNA translocase but not a processive helicase in vitro. The Journal ofbiological chemistry 282, 27076-27085 (2007); Ha et al (2008) supra; J.A. Ali, T. M. Lohman, Kinetic measurement of the step size of DNAunwinding by Escherichia coli UvrD helicase. Science (New York, N.Y.275, 377-380 (1997)). In order to investigate the processivity of Rep-X,we employed a dual optical tweezers assay (FIG. 3A; J. R. Moffitt etal., Intersubunit coordination in a homomeric ring ATPase. Nature 457,446-450 (2009)) that can monitor unwinding amplitudes and speeds overthousands of base pairs of DNA. The two traps held two streptavidinfunctionalized sub-micron sized polystyrene beads. The first was coatedwith 6-kbp dsDNA attached via a biotin on the blunt end and containing a3′ poly-dT ssDNA overhang on the other end ((dT)₁₀ (SEQ ID NO: 33),(dT)₁₅ (SEQ ID NO: 34), and (dT)₇₅ (SEQ ID NO: 35) see Example 7)). Theother bead was coated with Rep-X molecules via biotinylated antibodyagainst the hexa-histidine-tag (SEQ ID NO: 36). A laminar flow cell withtwo parallel streams of buffer was created for controlling theinitiation of the unwinding reaction (inset of FIG. 3B; L. R. Brewer, P.R. Bianco, Laminar flow cells for single-molecule studies of DNA-proteininteractions. Nature methods 5, 517-525 (2008)). When the two beads werebrought in proximity in the first laminar stream (Buffer C with 100 μMATP and 100 μM ATP-γS), a single Rep-X binding to the 3′ overhang of theDNA formed a tether between the two beads without initiating unwinding.When the tethered beads were moved to the second laminar stream (BufferC and 1 mM ATP), the DNA tether between the beads progressivelyshortened as the Rep-X monomer unwound and pulled the DNA. Unlessotherwise stated, SSB was added to the second laminar stream in order toprevent any subsequent interaction of unwound ssDNA with other Rep-X onthe bead surface. The optical tweezers experiments that were performedwithout SSB yielded the same Rep-X behavior (Example 7). By operatingthe trap under force feedback control, the tension was maintained on theDNA at 10-22 pN, as indicated. Additional controls and considerationsascertained that the observed activity stemmed from a single Rep-Xregardless of the 3′-tail length and inclusion/omission of SSB (Example7). Remarkably, 95% (38 out of 40) of the Rep-X-DNA complexes tetheredthrough a 3′-tail unwound the entire 6-kbp DNA in a processive manner(FIG. 3B, D) and the average pause-free speed was 136 bp/s (FIG. 3C). Incomparison, only 3% (2 out of 61 at 4 pN tension, none at higher forces)of wild type Rep and 7% (5 out of 70) of Rep-Y complexes displayed suchprocessive unwinding events (FIG. 3D). Rep-X may have even greaterprocessivity than 6-kbp, currently only limited by the length of the DNAused. The processive activity of a crosslinked Rep-X monomer shows theinnate potential of these helicases that can be harnessed viaconformational control.

The amount of force Rep-_(X) can generate during unwinding was evaluatedby performing measurements without the force feedback. Fixing trappositions led to a rapid build-up of force on the Rep-_(X) in theopposite direction of unwinding until the measurement was terminated dueto the breakage of connection between the two beads (FIG. 3E). Thehighest loads achieved in this experiment were not enough to stall thehelicase permanently. More detailed analysis showed that the pause freeunwinding rate of Rep-_(X) was not impeded by increasing loads up to thelimits of the linear regime of our trap (FIG. 3F), approximately 60 pN.These results suggest that the engineered Rep-X is the strongesthelicase known to date (T. T. Perkins, H. W. Li, R. V. Dalal, J. Gelles,S. M. Block, Forward and reverse motion of single RecBCD molecules onDNA. Biophysical journal 86, 1640-1648 (2004); J. G. Yodh, M. Schlierf,T. Ha, Insight into helicase mechanism and function revealed throughsingle-molecule approaches. Quarterly reviews of biophysics 43, 185-217(2010))

In order to test if generation of a super active helicase can bereproduced for other helicases, thereby providing additional evidence ofthe conformational control mechanism, a PcrA-X helicase was engineeredfrom Bacillus stearothermophilus PcrA. The Rep mutant sequences used togenerate PcrA-_(X) include those nucleotide and amino acid sequencesidentified in Table 3.

TABLE 3 Amino Acid and Nucleotide Sequences for exemplary PcrA-_(X) proteinsPolypeptide/DNA/RNA (SEQ ID NO:_) 5′→3′(nucleotide sequence) N→C (amino acid sequence) Wild type PcrA helicaseATGAACTTTTTATCGGAACAGCTGCTCGCCCATTTAAACAAAG (gene sequence)AGCAACAAGAAGCCGTCAGGACGACGGAAGGCCCGCTGCTCA >gi|696477066:c17795-TTATGGCGGGGGCGGGAAGCGGGAAAACGCGGGTGTTGACGC 15621 GeobacillusACCGCATCGCCTATTTGATGGCGGAAAAGCATGTGGCGCCGT stearothermophilusGGAACATTTTGGCCATTACGTTTACGAACAAGGCGGCGCGCG ATCC 7953AAATGCGGGPACGTGTGCAGTCGCTCTTAGGTGGGGCGGCGG GBScontig0000036_2,AAGACGTCTGGATTTCGACGTTCCACTCGATGTGCGTCCGCAT whole genome shotgunTTTGCGCCGCGACATTGACCGCATCGGCATCAACCGCAATTTT sequence (SEQ ID NO: TCCATCCTTGATCCGACGGACCAGCTTTCAGTCATGAAAACGA 38)TTTTAAAAGAAAAAAACATAGACCCGAAAAAATTTGAGCCGCGGACGATTTTAGGCACGATCAGCGCGGCGAAAAACGAGCTGTTGCCTCCGGAGCAATTCGCGAAGCGGGCCTCGACGTATTACGAAAAAGTCGTCAGCGATGTGTATCAAGAATACCAACAGCGCCTGCTTCGCAATCATTCGCTCGATTTTGACGATTTGATCATGACGACGATCCAACTGTTTGACCGCGTGCCGGATGTGCTTCACTATTACCAATATAAGTTTCAGTACATTCATATTGATGAGTACCAGGATACGAACCGCGCTCAATATACGCTGGTCAAAAAGCTGGCGGAACGCTTTCAAAACATTTGCGCCGTCGGCGACGCCGACCAATCGATTTATCGGTGGCGCGGGGCGGACATCCAAAACATTTTGTCGTTCGAGCGCGACTATCCGAACGCAAAAGTCATTTTGCTTGAACAAAACTACCGCTCGACGAAGCGCATTTTGCAAGCGGCGAACGAAGTCATCGAGCATAACGTCAACCGGAAGCCGAAACGGCTTTGGACGGAAAACCCGGAAGGAAAGCCGATTCTTTATTATGAGGCGATGAACGAAGCGGACGAAGCGCAGTTTGTCGCTGGACGCATCCGCGAGGCGGTGGAGCGCGGCGAACGCCGCTACCGTGATTTTGCTGTCTTGTACCGGACGAACGCCCAGTCGCGTGTCATGGAGGAAATGTTGCTGAAAGCGAACATTCCGTATCAAATTGTCGGCGGCTTAAAGTTCTATGACCGGAAAGAAATTAAAGACATTCTCGCCTATTTGCGCGTCATTGCCAATCCGGACGATGATTTAAGCTTGCTTCGCATCATTAACGTGCCAAAACGCGGCATTGGCGCCTCGACGATCGACAAACTCGTCCGCTATGCAGCCGATCATGAGCTGTCCTTGTTTGAGGCGCTCGGCGAGCTAGAGATGATCGGGCTTGGCGCCAAAGCGGCCGGGGCGCTCGCCGCGTICCGCAGCCAGCTCGAGCAATGGACACAGCTGCAAGAATACGTCTCCGTCACCGAACTCGTCGAAGAAGTGCTCGACAAATCGGGCTACCGCGAGATGCTCAAGGCGGAGCGGACGATTGAAGCACAAAGCCGGCTCGAGAACTTGGATGAGTTTTTGTCGGTGACGAAGCATTTTGAAAATGTGAGCGACGATAAATCGCTCATCGCCTTTTTAACCGACTTGGCGCTCATTTCCGATTTGGACGAGCTGAACGGGACGGAACAGGCCGCTGAAGGAGATGCCCGTCATGTTGATGACGTTGCATGCCGCCAAAGGGCTCGAGTTTCCGGTCGTCTTTTTGATCGGCATGGAAGAAGGCATTTTCCCGCACAACCGCTCTCTCGAGGATGACGATGAGATGGAAGAAGAACGGCGGCTGGCGTACGTCGGCATCACCCGCGCGGAGGAAGAACTTGTGCTGACGAGCGCGCAAATGCGGACGTTGTTTGGCAACATCCAAATGAACCCGCCGTCGCGCTTTTTGAATGAAATTCCGGCGCATTTGCTTGAGACAGCCTCGCGCCGCCAAGCGGGCGCCTCCCGCCCGGCCGTTTCGCGCCCGCAGGCAAGCGGCGCCGTGGGATCGTGGAAAGTCGGCGATCGGGCGAATCACCGGAAATGGGGCATCGGCACCGTCGTCAGCGTCCGCGGCGGCGGCGACGACCAAGAGCTCGACATCGCCTTCCCGAGCCCGATCGGCATTAAACGGTTGCTTGCCAAATTTGCGCC GATTGAGAAAGTGTAGWild type PcrA helicase MNFLSEQLLAHLNKEQQEAVRTTEGPLLIMAGAGSGKTRVLTHR(amino acid sequence)IAYLMAEKHVAPWNILAITFTNKAAREMRERVQSLLGGAAEDV >gi|696477065|ref|WISTHSMCVRILRRDIDRIGINRNFSILDPTDQLSVMKTILKEKNI WP_033016687.1 ATP-DPKKFEPRTILGTISAAKNELLPPEQFAKRASTYYEKVVSDVYQE dependent DNA helicaseYQQRLLRNHSLDFDDLIMTTIQLFDRVPDVLHYYQYKYFQYIHIDE PcrA [GeobaciilusYQDTNRAQYTLVKKLAERFQNICAVGDADQSIYRWRGADIQNIL stearothermophilus]SFERDYPNAKVILLEQNYRSTKRILQAANEVIEHNVNRKPKRLWT (SEQ ID NO: 39)ENPEGKPILYYEAMNEADEAQFVAGRIREAVERGERRYRDFAVLYRTNAQSRVMEEMLLKANIPYQIVGGLKFYDRKEIKDILAYLRVIANPDDDLSLLRIINVPKRGIGASTIDKLVRYAADHELSLFEALGELEMIGLGAKAAGALAAFRSQLEQWTQLQEYVSVTELVEEVLDKSGYREMLKAERTIEAQSRLENLDEFLSVTKHFENVSDDKSLIAFLTDLALISDLDELNGTEQAAEGDAVMLMTLHAAKGLEFPVVFLIGMEEGIFPHNRSLEDDDEMEEERRLAYVGITRAEEELVLTSAQMRTLFGNIQMNPPSRFLNEIPAHLLETASRRQAGASRPAVSRPQASGAVGSWKVGDRANHRKWGIGTVVSVRGGGDDQELDIAFPSPIGIKKL LAKFAPIEKVPcrA-_(X) polypeptide¹ MNFLSEQLLAHLNKEQQEAVRTTEGPLLIMAGAGSGKTRVLTHR(SEQ ID NO: 9) IAYLMAEKHVAPWNILAITFTNKAAREMRERVQSLLGGAAEDVWISTFHSMAVRILRRDIDRIGINRNFSILDPTDQLSVMKTILKEKNIDPKKFEPRTILGTISAAKNELLPPEQFAKRASTYYEKVVSDVYQEYQQRLLRCHSLDFDDLIMTTIQLFDRVPDVLHYYQYKFQYIHIDEYQDTNRAQYTLVKKLAERFQNIAAVGDADQSIYRWRGADIQNILSFERDYPNAKVILLEQNYRSTKRILQAANEVIEHNVNRKPKRLWTENPEGKPILYYEAMNEADEAQFVAGRIREAVERGERRYRDFAVLYRTNAQSRVMEEMLLKANIPYQIVGGVKFYDRKEIKDILAYLRVIANPDDDCSLLRIINVPKRGIGASTIDKLVRYAADHELSLFEALGELEMIGLGAKAAGALAAFRSQLEQWTQLQEYVSVTELVEEVLDKSGYREMLKAERTIEAQSRLENLDEFLSVTKHFENVSDDKSLIAFLTDLALISDLDELNGTEQAAEGDAVMLMTLHAAKGLEFPVVFLIGMEEGIFPHNRSLEDDDEMEEERRLAYVGITRAEEELVLTSAQMRTLFGNIQMNPPSRFLNEIPAHLLETASRRQAGASRPAVSKPQASGAVGSWKVGDRANHRKWGIGTVVSVRGGGDDQELDIAFPSPIGIKRL LAKFAPIEKV PcrA-_(X) DNA²ATGAACTTTTTATCGGAACAGCTGCTCGCCCATTTAAACAAAG (SEQ ID NO: 10)AGCAACAAGAAGCCGTCAGGACGACGGAAGGCCCGCTGcrcATTATGGCGGGGGCGGGAAGCGGGAAAACGCGGGTGTTGACGCACCGCATCGCCTATTTGATGGCGGAAAAGCATGTGGCGCCGTGGAACATTTTGGCCATTACGTTTACGAACAAGGCGGCGCGCGAAATGCGGGAACGTGTGCAGTCGCTCTTAGGTGGGGCGGCGGAAGACGTCTGGATTTCGACGTTCCACTCGATGGCCGTCCGCATTTTGCGCCGCGACATTGACCGCATCGGCATCAACCGCAATTTTTCCATCCTTGATCCGACGGACCAGCTTTCAGTCATGAAAACGATTTTAAAAGAAAAAAACATAGACCCGAAAAAATTTGAGCCGCGGACGATTTTAGGCACGACAGCGCGGCGAAAAACGAGCTGTTGCCTCCGGAGCAATTCGCGAAGCGGGCCTCGACGTATTACGAAAAAGTCGTCAGCGATGTGTATCAAGAATACCAACAGCGCCTGCTTCGCTGTCATTCGCTCGATTTTGACGATTTGATCATGACGACGATCCAACTGTTTGACCGCGTGCCGGATGTGCTTCACTATTACCAATATAAGTTTCAGTACATTCATATTGATGAGTACCAGGATACGAACCGCGCTCAATATACGCTGGTCAAAAAGCTGGCGGAACGCTTCAAAACATTGCCGCCGTCGGCGACGCCGACCAATCGATTTATCGGTGGCGCGGGGCGGACATCCAAAACATTTTGTCGTTCGAGCGCGACTATCCGAACGCAAAAGTCATTTTGCTTGAACAAAACTACCGCTCGACGAAGCGCATTTTGCAAGCGGCGAACGAAGTCATCGAGCATAACGTCAACCGGAAGCCGAAACGGCTTTGGACGGAAAACCCGGAAGGAAAGCCGATTCTTTATTATGAGGCGATGAACGAAGCGGACGAAGCGCAGTTTGTCGCTGGACGCATCCGCGAGGCGGTGGAGCGCGGCGAACGCCGCTACCGTGATTTTGCTGTCTTGTACCGGACGAACGCCCAGTCGCGTGTCATGGAGGAAATGTTGCTGAAAGCGAACATTCCGTATCAAATTGTCGGCGGCGTAAAGTTCTATGACCGGAAAGAAATTAAAGACATTCTCGCCTATTTGCGCGTCATTGCCAATCCGGACGATGATTGCAGCTTGCTTCGCATCATTAACGTGCCAAAACGCGGCATTGGCGCCTCGACGATCGACAAACTCGTCCGCTATGCAGCCGATCATGAGCTGTCCTTGTTTGAGGCGCTCGGCGAGCTAGAGATGATCGGGCTTGGCGCCAAAGCGGCCGGGGCGCTCGCCGCGTTCCGCAGCCAGCTCGAGCAATGGACACAGCTGCAAGAATACGTCTCCGTCACCGAACTCGTCGAAGAAGTGCTCGACAAATCGGGCTACCGCGAGATGCTCAAGGCGGAGCGGACGATTGAAGCACAAAGCCGGCTCGAGAACTTGGATGAGTTTTTGTCGGTGACGAAGCATTTTGAAAATGTGAGCGACGATAAATCGCTCATCGCCTTTTTAACCGACTTGGCGCTCATTTCCGATTTGGACGAGCTGAACGGGACGGAACAGGCCGCTGAAGGAGATGCCGTCATGTTGATGACGTTGCATGCCGCCAAAGGGCTCGAGTTTCCGGTCGTCTTTTTGATCGGCATGGAAGAAGGCATTTTCCCGCACAACCGCTCTCTCGAGGATGACGATGAGATGGAAGAAGAACGGCGGCTGGCGTACGTCGGCATCACCCGCGCGGAGGAAGAACTTGTGCTGACGAGCGCGCAAATGCGGACGTTGTTTGGCAACATCCAAATGAACCCGCCGTCGCGCTTTTTGAATGAAATTCCGGCGCATTTGCTTGAGACAGCCTCGCGCCGCCAAGCGGGCGCCTCCCGCCCGGCCGTTTCGCGCCCGCAGGCAAGCGGCGCCGTGGGATCGTGGAAAGTCGGCGATCGGGCGAATCACCGGAAATGGCUGCATCGGCACCGTCGTCAGCGTCCGCGGCGGCGGCGACGACCAAGAGCTCGACATCGCCTTCCCGAGCCCGATCGGUATTAAACGGTTGCTTGCCAAATTTGCGCC GATTGAGAAAGTGTAGPcrA-_(X) RNA³ AUGAACUUUUUAUCGGAACAGCUGCUCGCCCAUUUAAACAA (SEQ ID NO: 11)AGAGCAACAAGAAGCCGUCAGGACGACGGAAGGCCCGCUGCUCAUUAUGGCGGGGGCGGGAAGCGGGAAAACGCGGGUGUUGACGCACCGCAUCGCCUAUUUGAUGGCGGAAAAGCAUGUGGCGCCGUGGAACAUUUUGGCCAUUACGUUUACGAACAAGGCGGCGCGCGAAAUGCGGGAACGUGUGCAGUCGCUCUUAGGUGGGGCGGCGGAAGACGUCUGGAUUUCGACGUUCCACUCGAUGGCCGUCCGCAUUUUGCGCCGCGACAUUGACCGCAUCGGCAUCAACCGCAAUUUUUCCAUCCUUGAUCCGACGGACCAGCUUUCAGUCAUGAAAACGAUUUUAAAAGAAAAAAACAUAGACCCGAAAAAAUUUGAGCCGCGGACGAUUUUAGGCACGAUCAGCGCGGCGAAAAACGAGCUGUUGCCUCCGGAGCAAUUCGCGAAGCGGGCCUCGACGUAUUACGAAAAAGUCGUCAGCGAUGUGUAUCAAGAAUACCAACAGCGCCUGCUUCGCUGUCAUUCGCUCGAUUUUGACGAUUUGAUCAUGACGACGAUCCAACUGUUUGACCGCGUGCCGGAUGUGCUUCACUAUUACCAAUNUAAGUUUCAGUACAUUCAUAUUGAUGAGUACCAGGAUACGAACCGCGCUCAAUAUACGCUGGUCAAAAAGCUGGCGGAACGCUUUCAAAACAUUGCCGCCGUCGGCGACGCCGACCAAUCGAUUUAUCGGUGGCGCGGGGCGGACAUCCAAAACAUUUGUCGUUCGAGCGCGACUAUCCGAACGCAAAAGUCAUUUUGCUUGAACAAAACUACCGCUCGACGAAGCGCAUUUUGCAAGCGGCGAACGAAGUCAUCGAGCAUAACGUCAACCGGAAGCCGAAACGGCUUUGGACGGAAAACCCGGAAGGAAAGCCGAUUCUUUAUUAUGAGGCGAUGAACGAAGCGGACGAAGCGCAGUUUGUCGCUGGACGCAUCCGCGAGGCGGUGGAGCGCGGCGAACGCCGCUACCGUGAUUUUGCUGUCUUGUACCGGACGAACGCCCAGUCGCGUGUCAUGGAGGAAAUGUUGCUGAAAGCGAACAUUCCGUAUCAAAUUGUCGGCGGCGUAAAGUUCUAUGACCGGAAAGAAAUUAAAGACAUUCUCGCCUAUUUGCGCGUCAUUGCCAAUCCGGACGAUGAUUGCAGCUUGCUUCGCAUCAUUAACGUGCCAAAACGCGGCAUUGGCGCCUCGACGAUCGACAAACUCGUCCGCUAUGCAGCCGAUCAUGAGCUGUCCUUGUUUGAGGCGCUCGGCGAGCUAGAGAUGAUCGGGCUUGGCGCCAAAGCGGCCGGGGCGCUCGCCGCGUUCCGCAGCCAGCUCGAGCAAUGGACACAGCUGCAAGAAUACGUCUCCGUCACCGAACUCGUCGAAGAAGUGCUCGACAAAUCGGGCUACCGCGAGAUGCUCAAGGCGGAGCGGACGAUUGAAGCACAAAGCCGGCUCGAGAACUUGGAUGAGUUUUUGUCGGUGACGAAGCAUUUUGAAAAUGUGAGCGACGAUAAAUCGCUCAUCGCCUUUUUAACCGACUUGGCGCUCAUUUCCGAUUUGGACGAGCUGAACGGGACGGAACAGGCCGCUGAAGGAGAUGCCGUCAUGUUGAUGACGUUGCAUGCCGCCAAAGGGCUCGAGUUUCCGGUCGUCUUUUUGAUCGGCAUGGAAGAAGGCAUUUUCCCGCACAACCGCUCUCUCGAGGAUGACGAUGAGAUGGAAGAAGAACGGCGGCUGGCGUACGUCGGCAUCACCCGCGCGGAGGAAGAACUUGUGCUGACGAGCGCGCAAAUGCGGACGUUGUUUGGCAACAUCCAAAUGAACCCGCCGUCGCGCUUUUUGAAUGAAAUUCCGGCGCAUUUGCUUGAGACAGCCUCGCGCCGCCAAGCGGGCGCCUCCCGCCCGGCCGUUUCGCGCCCGCAGUCAAGCGGCGCCGUGGGAUCGUGGAAAGUCGGCGAUCGGGCGAAUCACCGGAAAUGUGGCAUCGGCACCGUCGUCAGCGUCCGCGGCGGCGGCGACGACCAAGAGCUCGACAUCGCCUUCCCGAGCCCGAUCGGCAUUAAACGGUUGCUUGCCAAAUUUGCGCCGAUUGAGAAAGU GUAG PerA-_(X) polypeptide⁴SEQ ID NO: 9 and formula no 1 in Table 2  (SEQ ID NO: 12)(1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dime). ¹This PcrA mutant encodesmutations removing natural cysteine residues found in the wild-type PcrAand include further amino acid mutations to facilitate intramolecularcrosslinking to an intramolecular crosslinking agent to generate thePcrA-_(X) polypeptide. ²The DNA sequence corresponds to the open readingframe that encodes the polypeptide of SEQ ID NO: 9. ³The RNA sequencecorresponds to the open reading frame that encodes the polypeptide ofSEQ ID NO: 9. ⁴The PcrA-_(X) polypeptide closed form monomer followingreaction of PcrA-_(X) polypeptide (SEQ ID NO: 9) with an intramolecularcrosslinking agent.

Exemplary intramolecular crosslinking agents suitable for generatingversions of PcrA-_(X) include those identified in Table 2. Methods andconditions for generating intramolecular crosslink formation in proteinsare well known in the art for these types of intramolecular crosslinkingagents, and such methods and conditions are applicable to the PcrAhelicases of this disclosure.

Mutations involved replacing two highly conserved Cys residues in thishelicase (FIG. 4A, B) which reduced the apparent ssDNA-dependent ATPaseactivity from approximately 40 ATP/s (wild type) to 5 ATP/s. Uponcrosslinking in the closed form, PcrA-_(X) retained the low ATPaseactivity (4.3 ATP/s), but exhibited an enhanced helicase activity incomparison to both the wild type and non-crosslinked mutant in ensemblereactions (FIG. 5A, B). smFRET experiments showed that PcrA-X monomerscan unwind 39% (228 out of 578) of the 18-bp dsDNA they bind compared toonly 4% (26 out of 617) for wild type PcrA (FIG. 6A-C). In the opticaltweezers assay, PcrA-X monomers, like Rep-X, were capable ofprocessively unwinding of 1-6 kbp long DNA, albeit at a much lower rate(2-15 bp/s, FIG. 6D) whereas no PcrA molecule (0 out of 51) was able todo the same (FIG. 6E). Despite the impaired activity levels of the PcrAmutant, conversion to PcrA-X made its monomers into highly processivehelicases, thus indicating a general mechanism of conformational controlfor this class of helicases.

Strong helicase activity of Rep-X and PcrA-X raises the possibility thatthe cellular partners of Rep or PcrA may switch on the powerfulunwinding activity intrinsic to these enzymes by constraining them inthe closed conformation. One such partner of PcrA is RepD, a plasmidreplication initiator protein from Staphylococcus aureus that recognizesand forms a covalent adduct with the oriD sequence of the plasmid, andthen recruits PcrA for highly processive unwinding (A. F. Slatter, C. D.Thomas, M. R. Webb, PcrA helicase tightly couples ATP hydrolysis tounwinding double-stranded DNA, modulated by the initiator protein forplasmid replication, RepD. Biochemistry 48, 6326-6334 (2009); W. Zhanget al., Directional loading and stimulation of PcrA helicase by thereplication initiator protein RepD. Journal of molecular biology 371,336-348 (2007); C. Machon et al., RepD-mediated recruitment of PcrAhelicase at the Staphylococcus aureus pC221 plasmid replication origin,oriD. Nucleic acids research 38, 1874-1888 (2010)). Based on the similarresults from PcrA-X and the homologous E. coli counterpart Rep-X, butnot Rep-Y, we hypothesized that the RepD-induced PcrA activityenhancement is in fact the result of the conformational constraint ofthe helicase in the PcrA-X-like closed form. To test this prediction, weprepared an oriD DNA-RepD adduct, and measured the intramolecularconformation of PcrA bound to this adduct. We used a double cysteinemutant of PcrA, PcrA-DM1, stochastically labeled with a mixture of donorand acceptor fluorophores that would be expected to generate highE_(FRET) in the closed form and low E_(FRET) in the open form (J. Parket al., PcrA helicase dismantles RecA filaments by reeling in DNA inuniform steps. Cell 142, 544-555 (2010); (FIG. 6F). The E_(FRET)distributions of PcrA-DM1 bound to the oriD DNA-RepD adduct and the oriDDNA alone are shown in FIG. 6F. Only the PcrA-DM1 molecules with afluorescence active Cy3-Cy5 pair were included in the analysis. Theresults revealed that the presence of RepD indeed biases PcrA toward theclosed high E_(FRET) conformation. Without the invention being limitedto any particular mechanism, the regulation mechanism of this class ofhelicases may involve in vivo partner proteins that constrain theconformation of 2B subdomain to the closed form to activate itsfunction.

The basis for constraining Rep and PcrA into the closed form thatconverts an enzyme with undetectable unwinding activity to a superhelicase is unknown. One possibility is that the intrinsic unwindingactivity itself requires the closed form, for example via thetorque-wrench mechanism proposed for UvrD (J. Y. Lee, W. Yang, UvrDhelicase unwinds DNA one base pair at a time by a two-part power stroke.Cell 127, 1349-1360 (2006)). Another possibility is that the open forminhibits helicase function and crosslinking to the closed form preventsthis inhibitory mechanism. Without the invention being limited to anyparticular theory of operation, we prefer the latter for the followingreasons. First, Rep-Y crosslinked in the open form does unwind DNA aswell as the wild type when the protein is at high concentrations inexcess of DNA (FIG. 1E). Therefore, the closed form per se is notabsolutely required for unwinding activity. Second, using ultra-highresolution optical tweezers combined with smFRET capability, we foundthat UvrD assumes the closed conformation when it unwinds DNA but afterit unwinds about 10 bp it switches to the open conformation and rewindsthe DNA likely after strand switching. Therefore, we suggest that Rep-Xbecomes a highly processive super-helicase because crosslinking preventsthe open conformation required for strand-switching and rewinding thathave been observed for UvrD (M. N. Dessinges, T. Lionnet, X. G. Xi, D.Bensimon, V. Croquette, Single-molecule assay reveals strand switchingand enhanced processivity of UvrD. Proc. Natl. Acad. Sci., U.S.A. 101,6439-6444 (2004)) and BLM (J. G. Yodh, B. C. Stevens, R. Kanagaraj, P.Janscak, T. Ha, BLM helicase measures DNA unwound before switchingstrands and hRPA promotes unwinding reinitiation. The EMBO journal 28,405-416 (2009)). The enhancement of unwinding activity via the deletionof 2B domain in Rep (W. Cheng et al., The 2B domain of the Escherichiacoli Rep protein is not required for DNA helicase activity. Proc. Natl.Acad. Sci., U.S.A. 99, 16006-16011 (2002)) may also be due to inhibitionof strand switching (M. J. Comstock, K. D. Whitley, H. Jia, T. M.Lohman, T. Ha and Y. R. Chemla, “Direct observation ofstructure-function relationship in a nucleic acid processing enzyme,”Science 348: 352-354 (2015).

Most conformational control of protein functions demonstrated so farfirst locks the naturally active protein to an artificially inhibitedconformation so that additional controls imposed by researchers can beused to recover the original activity (B. Choi, G. Zocchi, Y. Wu, S.Chan, L. Jeanne Perry, Allosteric control through mechanical tension.Phy Rev Lett 95, 078102 (2005); M. Tomishige, R. D. Vale, Controllingkinesin by reversible disulfide cross-linking. Identifying themotility-producing conformational change. J Cell Biol 151, 1081-1092(2000); D. M. Veine, K. Ohnishi, C. H. Williams, Jr., Thioredoxinreductase from Escherichia coli: evidence of restriction to a singleconformation upon formation of a crosslink between engineered cysteines.Protein science: a publication of the Protein Society 7, 369-375 (1998);B. X. Huang H. Y. Kim, Interdomain conformational changes in Aktactivation revealed by chemical cross-linking and tandem massspectrometry. Mol Cell Proteomics 5, 1045-1053 (2006)). Our work isinnovative and unique in that we found a conformational control thatactivates a naturally inhibited unwinding function, and the resultingenzyme is a super-helicase that has unprecedentedly high processivityfor a single motor helicase. RecBCD, another SF-1 helicase, hassimilarly high processivity but contains two motors and associatednucleases. Moreover it is known to backslide at opposing forces below 10pN whereas Rep-X can be active against forces as high as 60 pN (Perkinset al (2004) supra). This super helicase with high processivity and hightolerance against load without nuclease activities may also be usefulfor biotechnological applications such as single molecule nanoporesequencing (D. Branton et al., The potential and challenges of nanoporesequencing. Nature biotechnology 26, 1146-1153 (2008); A. H. Laszlo etal., Decoding long nanopore sequencing reads of natural DNA. Naturebiotechnology, (2014)) and isothermal DNA amplification (M. Vincent, Y.Xu, H. Kong, Helicase-dependent isothermal DNA amplification. EMBOreports 5, 795-800 (2004).

In this regard, one type of isothermal DNA amplification for Which thesesuper helicases have application include helicase dependentamplification. Referring to FIG. 8, the helicase dependent amplificationcan be characterized in three steps. In step 1, DNA helicase (104)contacts a double-stranded DNA (101) to unwind the first and secondsingle strands (102 and 103) to provide the ability of first and secondoligonucleotide primers (105 and 106) hybridize to the first and secondsingle strands (102 and 103), respectively. In step 2: DNA-dependent DNApolymerases (107) bind to the 3′-termini of the first and secondoligonucleotide primers (105 and 106) to initiate chain elongation ofnew strands (108 and 109). In step 3, continued DNA polymerizationresults in DNA amplification and formation of new double-stranded DNA(110 and 111).

Nucleic Acid Amplification

In certain exemplary embodiments, methods for amplifying nucleic acidsequences are provided. Exemplary methods for amplifying nucleic acidsinclude the polymerase chain reaction (PCR) (see, e.g., Mullis et al.(1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263 and Cleary etal. (2004) Nature Methods 1:241; and U.S. Pat. Nos. 4,683,195 and4,683,202), anchor PCR, RACE PCR, ligation chain reaction (LCR) (see,e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:360-364), self-sustainedsequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.U.S.A. 87:1874), transcriptional amplification system (Kwoh et al.(1989) Proc. Natl. Acad. Sci. U.S.A. 86:1173), Q-Beta Replicase (Lizardiet at (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000)J. Biol. Chem. 275:2619; and Williams et al. (2002) J. Biol. Chem.277:7790), the amplification methods described in U.S. Pat. Nos.6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199,isothermal amplification (e.g., rolling circle amplification (RCA),hyperbranched rolling circle amplification (HRCA), strand displacementamplification (SDA), helicase-dependent amplification (HDA), PWGA, orany other nucleic acid amplification method using techniques well knownto those of skill in the art.

“Polymerase chain reaction,” or “PCR,” refers to a reaction for the invitro amplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions of the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites, and(iii) extending the primers by a nucleic acid polymerase in the presenceof nucleoside triphosphates. Usually, the reaction is cycled throughdifferent temperatures optimized for each step in a thermal cyclerinstrument. Particular temperatures, durations at each step, and ratesof change between steps depend on many factors well-known to those ofordinary skill in the art, e.g., exemplified by the references:McPherson et al., editors, PCR: A Practical Approach and PCR2: APractical Approach (IRL Press, Oxford, 1991 and 1995, respectively). Forexample, in a conventional PCR using Taq DNA polymerase, a doublestranded target nucleic acid may be denatured at a temperature greaterthan 90° C., primers annealed at a temperature in the range 50-75° C.,and primers extended at a temperature in the range 72-78° C.

The term “PCR” encompasses derivative forms of the reaction, includingbut not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR,multiplexed PCR, assembly PCR and the like. Reaction volumes range froma few hundred nanoliters, e.g., 200 nL, to a few hundred microliters,e.g., 200 microliters. “Reverse transcription PCR,” or “RT-PCR,” means aPCR that is preceded by a reverse transcription reaction that converts atarget RNA to a complementary single stranded DNA, which is thenamplified, e.g., Tecott et al., U.S. Pat. No. 5,168,038. “Real-time PCR”means a PCR for which the amount of reaction product, i.e., amplicon, ismonitored as the reaction proceeds. There are many forms of real-timePCR that differ mainly in the detection chemistries used for monitoringthe reaction product, e.g., Gelfand et al., U.S. Pat. No. 5,210,015(“Taqman”); Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627(intercalating dyes); Tyagi et al., U.S. Pat. No. 5,925,517 (molecularbeacons). Detection chemistries for real-time PCR are reviewed in Mackayet al., Nucleic Acids Research, 30:1292-1305 (2002). “Nested PCR” meansa two-stage PCR wherein the amplicon of a first PCR becomes the samplefor a second PCR using a new set of primers, at least one of which bindsto an interior location of the first amplicon. As used herein, “initialprimers” in reference to a nested amplification reaction mean theprimers used to generate a first amplicon, and “secondary primers” meanthe one or more primers used to generate a second, or nested, amplicon.“Multiplexed PCR” means a PCR wherein multiple target sequences (or asingle target sequence and one or more reference sequences) aresimultaneously carried out in the same reaction mixture, e.g. Bernard etal. (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR).Usually, distinct sets of primers are employed for each sequence beingamplified. “Quantitative PCR” means a PCR designed to measure theabundance of one or more specific target sequences in a sample orspecimen. Techniques for quantitative PCR are well-known to those ofordinary skill in the art, as exemplified in the following references:Freeman et al., Biotechniques, 26:112-126 (1999); Becker-Andre et al.,Nucleic Acids Research, 17:9437-9447 (1989); Zimmerman et al.,Biotechniques, 21:268-279 (1996); Diviacco et al., Gene, 122:3013-3020(1992); Becker-Andre et al., Nucleic Acids Research, 17:9437-9446(1989); and the like.

In one aspect of the invention, a method of performing isothermal DNAamplification is provided. The method can includes two steps. The firststep includes forming a mixture. The mixture includes a double-strandedDNA template having a first strand and a second strand; aconformationally-constrained helicase; a DNA-dependent DNA polymerase; afirst oligonucleotide primer complementary to a portion of the firststrand; a second oligonucleotide primer complementary to a portion ofthe second strand; and an amplification buffer cocktail. The second stepincludes incubating the mixture at a temperature compatible foractivating the conformationally-constrained helicase and DNA-dependentDNA polymerase. In some embodiments of this aspect, theconformationally-constrained helicase is selected from SEQ ID NOs: 4 and12.

Nucleic Acid Sequencing

In certain exemplary embodiments, methods of determining the sequenceidentities of nucleic acid sequences are provided. Determination of thesequence of a nucleic acid sequence of interest can be performed usingvariety of sequencing methods known in the art including, but notlimited to, sequencing by hybridization (SBH), sequencing by ligation(SBL), quantitative incremental fluorescent nucleotide additionsequencing (QIFNAS), stepwise ligation and cleavage, fluorescenceresonance energy transfer (FRET), molecular beacons, TaqMan reporterprobe digestion, pyrosequencing, fluorescent in situ sequencing(FISSEQ), FISSEQ beads (U.S. Pat. No. 7,425,431), wobble sequencing(PCT/US05/27695), multiplex sequencing (U.S. 2008/0269068; Porreca et al(2007) Nat. Methods 4:931), polymerized colony (POLONY) sequencing (U.S.Pat. Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT/US05/06425),nanogrid rolling circle sequencing (ROLONY) (U.S. 2009/0018024),nanopore sequencing (using platforms such as those from Agilent, Oxford,Sequenom, Noblegen, NABsys, Genia), allele-specific oligo ligationassays (e.g., oligo ligation assay (OLA), single template molecule OLAusing a ligated linear probe and a rolling circle amplification (RCA)readout, ligated padlock probes, and/or single template molecule OLAusing a ligated circular padlock probe and a rolling circleamplification (RCA) readout) and the like. High-throughput sequencingmethods, e.g., on cyclic array sequencing using platforms such as Roche454, Illumina Solexa, ABI-SOLiD, ION Torrents, Complete Genomics,Pacific Bioscience, Helicos, Polonator platforms (Worldwide Web Site:Polonator.org), and the like, can also be utilized. High-throughputsequencing methods are described in U.S. 2010/0273164. A variety oflight-based sequencing technologies are known in the art (Landegren etat (1998) Genome Res. 8:769-76; Kwok (2000) Pharmocogenomics 1:95-100;and Shi (2001) Clin. Chem. 47:164-172).

In certain exemplary embodiments, the DNA-dependent DNA polymerase isselected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol ILarge Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNAPolymerase Large Fragment Bsu DNA Polymerase I Large Fragment, T4 DNAPolymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase and phi29DNA Polymerase. In some embodiments, the conformationally-constrainedhelicase includes a helicase selected from superfamily 1, wherein thehelicase has a first amino acid residue and a second amino acid reside,and wherein the first and second amino acid residues are in proximity.The conformationally-constrained helicase also includes a linker,wherein the linker comprises a first covalent bond with the first aminoacid residue and a second covalent bond with the second amino acidresidue. In some embodiments of this aspect, theconformationally-constrained helicase includes a crosslinked, closedform helicase monomer.

Expression of Helicase-_(X) Polypeptides

The nucleic acids encoding the Rep-_(X) and PcrA-_(X) polypeptides canbe adapted to suitable expression systems for producing the helicase_(X)polypeptides for helicase-_(X) production. For DNAs encodinghelicase_(X) genes, the representative genes can be operably-linked tosuitable expression vectors for expressing the proteins in bacterial,fungal, insect or other suitable expression host. For RNAs encodinghelicase-_(X) polypeptides, the representative RNAs can be engineeredfor enabling efficient expression in vitro of the polypeptides inextract lysates produced from bacterial, fungal, insect or othersuitable expression host sources. Such systems are well known in theart. Following expression, the helicase-_(X) polypeptides can bepurified by methods known in the art, including affinity-tagchromatography, SDS-PAGE, and size-exclusion chromatography, amongothers.

In certain exemplary embodiments, vectors such as, for example,expression vectors, containing a nucleic acid encoding one or morehelicase-_(X) polypeptides described herein are provided. As usedherein, the term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid,” which refers to a circular double stranded DNAloop into which additional DNA segments can be ligated. Another type ofvector is a viral vector, wherein additional DNA segments can be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)are integrated into the genome of a host cell upon introduction into thehost cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “expression vectors.” In general, expression vectors ofutility in recombinant DNA techniques are often in the form of plasmids.In the present specification, “plasmid” and “vector” can be usedinterchangeably. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

In certain exemplary embodiments, the recombinant expression vectorscomprise a nucleic acid sequence (e.g., a nucleic acid sequence encodingone or more helicase-_(X) polypeptides described herein) in a formsuitable for expression of the nucleic acid sequence in a host cell,which means that the recombinant expression vectors include one or moreregulatory sequences, selected on the basis of the host cells to be usedfor expression, which is operatively linked to the nucleic acid sequenceto be expressed. Within a recombinant expression vector, “operablylinked” is intended to mean that the nucleotide sequence encoding one ormore helicase-_(X) polypeptides is linked to the regulatory sequence(s)in a manner which allows for expression of the nucleotide sequence(e.g., in an in vitro transcription/translation system or in a host cellwhen the vector is introduced into the host cell). The term “regulatorysequence” is intended to include promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those which directconstitutive expression of a nucleotide sequence in many types of hostcells and those which direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the hostcell to be transformed, the level of expression of protein desired, andthe like. The expression vectors described herein can be introduced intohost cells to thereby produce proteins or portions thereof, includingfusion proteins or portions thereof, encoded by nucleic acids asdescribed herein (e.g., one or more helicase_(X) polypeptides).

Recombinant expression vectors of the invention can be designed forexpression of one or more encoding one or more helicase-_(X)polypeptides in prokaryotic or eukaryotic cells. For example, one ormore vectors encoding one or more helicase-_(X) polypeptides can beexpressed in bacterial cells such as E. coli, insect cells (e.g., usingbaculovirus expression vectors), yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40); pMAL (New EnglandBiolabs, Beverly, Mass.); and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

In another embodiment, the expression vector encoding one or morehelicase-_(X) polypeptides is a yeast expression vector. Examples ofvectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari,et. al., (1987) EMBO J. 6:229-234); pMFa (Kurjan and Herskowitz, (1982)Cell 30:933-943); pJRY88 (Schultz et al., (1987) Gene 54:113-123); pYES2(Invitrogen Corporation, San Diego, Calif.); and picZ (InvitrogenCorporation).

Alternatively, one or more helicase-_(X) polypeptides can be expressedin insect cells using baculovirus expression vectors. Baculovirusvectors available for expression of proteins in cultured insect cells(e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell.Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989)Virology 170:31-39).

In certain exemplary embodiments, a nucleic acid described herein isexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987)Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195).When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, adenovirus 2, cytomegalovirusand simian virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see Green M., and Sambrook; J.Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 2012.

In certain exemplary embodiments, host cells into which a recombinantexpression vector of the invention has been introduced are provided. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, oneor more helicase-_(X) polypeptides can be expressed in bacterial cellssuch as E. coli, viral cells such as retroviral cells, insect cells,yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) orCOS cells). Other suitable host cells are known to those skilled in theart.

Delivery of nucleic acids described herein (e.g., vector DNA) can be byany suitable method in the art. For example, delivery may be byinjection, gene gun, by application of the nucleic acid in a gel, oil,or cream, by electroporation, using lipid-based transfection reagents,or by any other suitable transfection method.

As used herein, the terms “transformation” and “transfection” areintended to refer to a variety of art-recognized techniques forintroducing foreign nucleic acid (e.g., DNA) into a host cell, includingcalcium phosphate or calcium chloride co-precipitation,DEAF-dextran-mediated transfection, lipofection (e.g., usingcommercially available reagents such as, for example, LIPOFECTIN™(Invitrogen Corp., San Diego, Calif.), LIPOFECTAMINE™ (Invitrogen),FUGENE™ (Roche Applied Science, Basel, Switzerland), JETPEI™(Polyplus-transfection Inc., New York, N.Y.), EFFECTENE™ (Qiagen,Valencia, Calif.), DREAMFECT™ (OZ Biosciences, France) and the like), orelectroporation (e.g., in vivo electroporation). Suitable methods fortransforming or transfecting host cells can be found in Green andSambrook, et al. (Molecular Cloning: A Laboratory Manual. 4th, ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2012), and other laboratory manuals.

Kits

In another aspect, kits are contemplated in this disclosure. Forexample, a kit for performing helicase dependent amplification isprovided. The kit can include a conformationally-constrained helicaseand an optional amplification buffer cocktail. Theconformationally-constrained helicase of the kit includes one or morehelicase_(X) polypeptides having a covalent linkage (e.g., reacted witha suitable intramolecular crosslinking agent) to form closed formhelicase-_(X) monomers having super helicase activity of the typedescribed for Rep-X and PcrA-X. In particular, theconformationally-constrained helicase can be generated form reacting SEQID NOs:4 and 9 with a suitable intramolecular crosslinking agent.Representative conformationally-constrained helicases include those ofSEQ ID NOs:4 and 12.

The kit can further include a DNA-dependent DNA polymerase. ExemplaryDNA-dependent DNA polymerases for inclusion in kit include a polymeraseselected from a group consisting of E. coli DNA Pol I, E. coli DNA Pol ILarge Fragment, Bst 2.0 DNA Polymerase, Bst DNA Polymerase, Bst DNAPolymerase Large Fragment, Bsu DNA Polymerase I Large Fragment, T4 DNAPolymerase, T7 DNA polymerase, PyroPhage® 3173 DNA Polymerase, phi29 DNAPolymerase and the like.

EXAMPLES Example 1. Mutagenesis and Purification of Protein

Preparation of pET expression plasmids containing cysteine-less rep(C18L, C43S, C167V, C178A, C612A) and pcrA (C96A/C247A) with N-terminalhexa-histidine-tags (SEQ ID NO: 36) were pet formed as describedpreviously (Park et al (2005) supra, I. Rasnik, S. Myong, W. Cheng T. M.Lohman, T. Ha, DNA-binding orientation and domain conformation of the E.coli rep helicase monomer bound to a partial duplex junction:single-molecule studies of fluorescently labeled enzymes. J. Mol. Biol.336, 395-408 (2004)). Site-directed mutations to introduce two Cysresidues for crosslinking (Rep-X: A178C/S400C, Cys178 is a nativecysteine in the wild type, Rep-Y: D127C/S494C, PcrA-X: N187C/L409C) weredone using QuikChange Lightning kit (Life Technologies, Inc.) andmutagenic primer oligonucleotides (Integrated DNA Technologies Inc.,Coralville, Iowa). Protein purifications were performed as describedpreviously (Park et al. (2005) supra; Rasnik et al (2004) supra).Catalytic activity levels of purified proteins as well as those of thecrosslinked samples were determined in a ssDNA-dependent ATPase activityassay using the Invitrogen EnzChek phosphate assay kit (LifeTechnologies Inc.), the oligonucleotide (dT)₄₅ (SEQ ID NO: 305) and 1 mMATP in buffer D (see ensemble FRET unwinding assay).

Wild type RepD from Staphylococcus aureus was purified as described in(Slatter et al. ((2009) supra; Zhang et al., (2007) supra) with thefollowing differences. A wt-RepD encoding pET11m-RepD plasmid wasconstructed for expression in B834 (pLysS). The gene sequence containedsilent mutations to introduce restriction sites for AgeI, PstI, SacI,and to modify the nick site (TCTAAT to TCGAAT) to prevent prematurecleavage by RepD during expression. An ammonium sulfate precipitatedpellet (from 0.5 L culture) was resuspended and run through seriallyconnected 5 ml Q-Sepharose (removed once the sample was through) and 5ml heparin-Sepharose cartridges connected in series (GE Healthcare), andeluted on an ÄKTA purifier 10 FPLC system.

Example 2. Intra-Crosslinking of Rep and PcrA

Dual-cysteine Rep mutants were incubated overnight at 4° C. with 2- to100-fold excess of bis-maleimide crosslinkers DTME (13 Å) and BMOE (8 Å)purchased from Thermo Fisher Scientific, Rockford, Ill. (FIG. 10).PcrA-X was crosslinked with DTME and BM(PEG)₂ (14.7 Å) from the samemanufacturer. Excess crosslinkers were removed by Bio-Rad P-30 desaltingcolumn. Crosslinked Rep-X, Rep-Y and PcrA-X samples were stored at −20°C. or −80° C. as described (Park et al. (2005) supra; Rasnik et al.(2004) supra). Data presented in this manuscript used BMOE (8 Å), butother crosslinkers of various lengths gave similar results. DTME is adi-sulfide containing crosslinker that we reduced with β-mercaptoethanol(β-ME) or tris(2-carboxyethyl) phosphine (TCEP) to revert thecrosslinked helicase to the non-crosslinked form for control purposes.

Crosslinking of the double Cys mutants with the his-maleimide linkershas the potential of producing covalently attached multimeric species,in addition to the intended internally crosslinked monomeric species.Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) candistinguish these species from the non-crosslinked monomers (I. L.Urbatsch et al., Cysteines 431 and 1074 are responsible for inhibitorydisulfide cross-linking between the two nucleotide-binding sites inhuman P-glycoprotein. J. Biol. Chem, 276, 26980-26987 (2001)). Here weshow a representative analysis of a crosslinked Rep-Y sample.Crosslinked Rep-X and Rep-Y produced three bands on a SDS polyacrylamidegel (FIG. 7A): a bottom band at ˜76 kDa that was the same as thenon-crosslinked Rep, a slightly retarded dominant middle band at ˜100kDa for Rep-Y and ˜90 kDa for Rep-X and a much more slowly migrated,very faint top band at ˜300 kDa. FIG. 7B shows three such bands of aRep-Y sample (lane Rep-Y) crosslinked with a cleavable di-sulfidecontaining crosslinker (DTME). The dominant middle band and the fainttop band were the crosslinked species because they disappeared uponcleavage of the crosslinker using beta-mercaptoethanol (β-ME) (laneRep-Y*). Relative shift between the middle bands of Rep-X and Rep-Y(FIG. 7A) was a strong indication of an internally crosslinked monomericspecies, because the denatured Rep-X and Rep-Y would be likely tomigrate at different rates due to the different size of peptide loopsintroduced by the internal crosslinker (denatured Rep-Y has a loop of368 amino acids (aa) whereas Rep-X loop is 223 aa long). In order toensure that the dominant middle band is not multimeric but is theintramolecularly crosslinked monomeric species, a Rep-Y sample wasfractionated according to molecular size on a Superdex 200 sizeexclusion chromatography (SEC) column controlled by an FPLC apparatus.Elution profiles of Rep-Y and non-crosslinked Rep are shown in the FIG.7C. Eluted fractions were analyzed on an SDS polyacrylamide gel (FIG.7D, lanes F1-F7). The multimeric species that was eluted in the earlySEC fractions (11-13 ml) displayed only the top band whereas thedominant middle band was eluted together with the non-crosslinked Repmonomer in the SEC analysis, showing that the middle band represents theintramolecularly crosslinked species and the top band is multimeric.After establishing that the intra-crosslinked protein shows up as aretarded band compared to the non-crosslinked form on the SDSpolyacrylamide gels (such as the Rep-Y data presented here), we usedthis assay to check the efficiency of crosslinking reactions for Rep-X,Rep-Y and PcrA-X (86%, 73% and 58% respectively for the samples used inthis manuscript). The Rep-Y form exhibited ATPase activity on par withnon-crosslinked Rep (FIG. 7E).

Example 3. Size Exclusion Chromatography and SDS-PAGE Analysis

Crosslinked Rep and PcrA samples were separated from multimericbyproducts using Superdex 200 grade 10/300GL or HiLoad 16/600 gelfiltration columns on an ÄKTA purifier 10 FPLC system. The crosslinkingefficiency was monitored by SDS-PAGE analysis on 7.5-10% Tris-glycinegels (Bio-Rad). As needed for gel analysis, reduction of samplescrosslinked with DTME was achieved by adding 5% (v/v) β-ME during theSDS denaturation step.

Example 4. Ensemble FRET Unwinding Assay

Multiple turnover ensemble unwinding kinetics was used to gauge theeffect of the mutations and conformational modifications to the helicaseactivity. We used an 18-bp FRET labeled DNA substrate with a 3′-(dT)₁₀overhang (SEQ ID NO: 33) (FIG. 1C), constructed by annealingcomplementary oligonucleotides DNA7 (Cy5-GCC TCG CTG CCG TCG CCA (SEQ IDNO: 40)) and amino-dT labeled DNA8 (TGG CGA CGG CAG CGA GGC-(T-Cy3)-T₁₀(SEQ ID NO: 41)). Alternatively, another similarly labeled 50-bp DNAwith 3′-(dT)₃₀ overhang (SEQ ID NO: 17) was also used. This constructwas made by annealing oligonucleotides DNA9 (Cy5-TCA ACT AGC AGT CAT AGGAGA AGT ATT AAC ATG CCT CGC TGC CGT CGC CA (SEQ ID NO: 42)) and amino-dTlabeled DNA10 (TG GCG ACG GCA GCG AGG CAT GTT AAT ACT TCT CCT ATG ACTGCT AGT TGA (T-Cy3) T₂₉ (SEQ ID NO: 43)). Unless otherwise stated, 5 nMensemble FRET DNA was mixed with 50 nM helicase in buffer D (10 mMTris-HCl [pH 8.0], 15 mM NaCl, 10 mM MgCl₂, 10% (v/v) glycerol, 0.1mg/ml BSA) and 1 mM ATP was added to start the unwinding reaction in aquartz cuvette. A Cary Eclipse fluorescence spectrophotometer was usedto measure the donor (I_(555nm)) and the acceptor signals (I_(667nm))under 545-nm excitation (5-nm slit, 2-10 Hz acquisition rate and600-900V photomultiplier voltage). Unwinding of the substrate wasmonitored by the decrease in ensemble E_(FRET) value, defined asE_(FRET-ensemble)=I_(667nm)/(I_(555nm)−I₀+I_(667nm)) where I₀ was thebaseline donor signal of unpaired Cy3 prior to addition of ATP.

Example 5. smFRET Unwinding and RepD-PcrA Interaction Assays

All smFRET experiments were conducted on a custom-built prism type TIRFmicroscopy stage with an Andor EMCCD camera as described in R. Roy, S.Hohng, T. Ha, A practical guide to single-molecule FRET. Nat Methods 5,507-516 (2008) and C. Joo, T. Ha, in Cold Spring Harb Protoc. (2012),vol. 2012. Reaction chambers were formed by quartz slides and glasscoverslips passivated with polyethyleneglycol (PEG) and 1% biotinylatedPEG (mPEG-SC and bio-PEG-SC, Laysan Bio, Arab, Ala.), followed by 5 minincubation with Neutravidin (Thermo Scientific, Newington, N.H.) forimmobilization of biotinylated molecules on the chamber surface asdescribed below.

For the smFRET unwinding experiments, the reaction chamber was firstincubated with biotinylated anti penta-histidine tag (SEQ ID NO: 44)antibody (Qiagen, Valencia, Calif.), followed by 10-30 min incubation ofHis₆-tagged (SEQ ID NO: 36) helicase sample (0.5-1 nM). The unwinding ofthe DNA was initiated by flowing 1 nM smFRET DNA and 1 mM ATP in thereaction buffer A (10 mM Tris-HCl [pH 8.0], 10 mM MgCl₂, 15 mM NaCl, 10%(v/v) glycerol, 1% (v/v) gloxy and 0.2% (w/v) glucose, an oxygenscavenging system (Y. Harada, K. Sakurada, T. Aoki, D. D. Thomas, T.Yanagida, Mechanochemical coupling in actomyosin energy transductionstudied by in vitro movement assay. J. Mol. Biol. 216, 49-68 (1990).)and 3-4 mM Trolox (T. Yanagida, M. Nakase, K. Nishiyama, F. Oosawa,Direct observation of motion of single F-actin filaments in the presenceof myosin. Nature 307, 58-60 (1984); I. Rasnik, S. A. McKinney, T. Ha,Nonblinking and long-lasting single-molecule fluorescence imaging. NatMethods 3, 891-893 (2006)). The smFRET DNA substrate was constructed byannealing the oligonucleotides DNA3 (Cy5-GCC TCG CTG CCG TCG CCA (SEQ IDNO: 40)) and DNA4 (Cy3-TGG CGA CGG CAG CGA GGC-T₂₀ (SEQ ID NO: 45)). ThePcrA-RepD interaction assay involved preparation of the RepD-oriD DNAadduct as described in Slatter et al (2009) supra. A biotinylated oriDDNA substrate was constructed by annealing oligonucleotides DNA1 (CTAATA GCC GGT TAA GTG GTA ATT TTT TTA CCA CCC AAA GCC TGA AGA GCT AAT CGTTCG G (SEQ ID NO: 46)) and DNA2 (biotin-CCG AAC GAT TAG CTC TTC AGG CTTTGG GTG GTA AAA: AA TTA CCA CTT T₁₅ (SEQ ID NO: 47)). In one chamber,only oriD DNA (50-100 pM) was immobilized on the surface. In a secondchamber the RepD-oriD DNA adduct was immobilized. 100-500 pM duallabeled PcrA-DM1 was injected into the chambers in buffer B (10 mM Tris[pH7.5], 10% glycerol, 15 mM NaCl, 50 mM KCl, 5 mM MgCl₂, 3.4 mM Trolox,1% (v/v) gloxy, 0.2% (w/v) glucose). Short movies of multiple chamberregions were recorded. Since the two Cys residues of PcrA-DM1 wererandomly labeled with Cy3-Cy5 mixture, each movie contained a briefinitial 633-nm laser excitation period to determine the molecules with afluorescent Cy5, followed by turning on the 532-nm laser for Cy3excitation. Only the PcrA-DM1 molecules with a colocalizeddonor-acceptor pair were factored in the E_(FRET) histograms.

smFRET signals were acquired by an Andor EMCCD camera operated with acustom software at 16-100-ms time resolution. E_(FRET) was calculated asdescribed in R. Roy, S. Hohng, T. Ha, A practical guide tosingle-molecule FRET. Nat Methods 5, 507-516 (2008). Unwinding periodswere measured as described in the text. The fraction of unwinding eventswas calculated as the proportion of the all DNA binding events thatdisplayed an E_(FRET) increase phase. Error bars were calculatedaccording to Clopper-Pearson binomial proportion confidence intervalmethod (C. J. Clopper, E. S. Pearson, The use of confidence or fiduciallimits illustrated in the case of the binomial. Biometrika 26, 404-413(1934)).

Example 6. Optical Tweezers Assay

The optical trap handle was a 6098-bp long DNA, amplified from λ-phageDNA and flanked by a 5′-biotin and a 3′-(dT)_(10,15,75) overhang (SEQ IDNOS 33-35, respectively) on the other end. First, a 5′-tailed 6083-bpfragment was amplified by the auto-sticky PCR reaction (J. Gal, R.Schnell, S. Szekeres, M. Kalman, Directional cloning of native PCRproducts with preformed sticky ends (autosticky PCR). Mol Gen. Genet.260, 569-573 (1999)) using primers P1 (biotin-GGC AGG GAT ATT CTG GCA(SEQ ID NO: 48)) and P2 (GAT CAG TGG ACA GA-abasic-A AGC CTG AAG AGC TAATCG TTC GG (SEQ ID NO: 49)). Subsequently the amplicon was annealed andligated with oligonucleotide DNA5 (TTC TGT CCA CTG ATC-(T)_(10,15,75)(SEQ ID NOS 50-52, respectively)) to create the 3′-overhang for theinitial helicase binding (10, 15 or 75-nt, as specified in figures). DNAbeads were prepared by adding biotinylated 6-kbp DNA to thestreptavidin-coated polystyrene beads (0.79 μm in diameter, Spherotech,Lake Forest, Ill.), and incubated at 25° C. for 30 min. Protein sampleswere pre-incubated with biotinylated anti penta-histag (SEQ ID NO: 44)antibody (Qiagen, Valencia, Calif.) on ice for 1 hour. One microliter ofthis mixture, 1 μl of streptavidin beads, and 8 μl buffer (100 mMTris-HCl [pH 7.5], 100 mM NaCl, 10% glycerol (v/v)) were mixed andincubated for 30 min on ice to make the protein coated beads. Reactionswere performed in laminar flow chambers that were designed and assembledas described in Z. Qi, R A. Pugh, M. Spies, Y. R. Chemla,Sequence-dependent base pair stepping dynamics in XPD helicaseunwinding. Elife (Cambridge) 2, e00334 (2013). Reaction buffer Cconsisted of 100 mM Tris pH 8.0, 15 mM NaCl, 10% (v/v) glycerol, 10 mMMgCl₂, and an oxygen scavenging system (100 μg/ml glucose oxidase, 20μg/ml catalase, and 4 mg/ml glucose) to reduce photo damage to thesample (M. P. Landry, P. M. McCall, Z. Qi, Y. R. Chemla,Characterization of photoactivated singlet oxygen damage insingle-molecule optical trap experiments. Biophysical journal 97,2128-2136 (2009)). The reaction chamber contained two laminar streams ofbuffer C with different ATP, ATP-γS and SSB concentrations as describedin the text. The dual-trap optical tweezers were set up and calibratedas described in (C. Bustamante, Y. R. Chemla, J. R. Moffitt,High-resolution dual-trap optical tweezers with differential detection.Single-molecule techniques: a laboratory manual (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2008); K. Berg-Sørensen, H.Flyvbjerg, Power spectrum analysis for optical tweezers. Review ofScientific Instruments 75, 594-612 (2004)). All measurements wererecorded at 100 Hz with a custom LabView software (8.2; Nationalinstruments, Austin, Tex.) and smoothed with a 100 Hz boxcar filter. Inthe “force-feedback” mode, unwinding was allowed to occur against aconstant force of 10-22 pN (as specified). The contour length of DNA wascalculated from the measured force and end-to-end extension of themolecule and using the worm-like chain model (persistence length of 53nm, stretch modulus of 1,200 pN and distance per base-pair of 0.34 nm).The velocity of DNA unwinding in the force feedback mode was determinedfrom a linear fit of the contour length of DNA in a sliding window of0.2 s (21 data points). Pauses longer than 0.2 s were removed and thenthe velocity was averaged in 1 s bins. Error for the fraction ofunwinding events per tether formation was calculated with theClopper-Pearson binomial proportion confidence interval method (Clopperet al. (1934) supra)).

The force dependence of Rep-X unwinding activity was measured in the“fixed-trap” mode, by stopping the force feedback. The force data (100Hz) was smoothed with a gaussian filter (by applying a 33-Hz movingaverage filter 10 times). Paused regions (velocity <10 bp/s) wereremoved. The pause-free unwinding velocities were calculated andnormalized by the velocity at 20 pN for each molecule, and binnedagainst the dynamic force values up to 60 pN to create the V_(norm) vs.F plot (FIG. 3F). We previously found that the force response of ourtrap was linear against bead displacements up to 72 nm (determined in aseparate experiment measuring where the force vs. extension curve ofdsDNA started to deviate from the theoretical worm like chain. At a trapstiffness of 0.167 pN/nm, the deviation occurred above 12 pN). Hence wecalculated the maximum reliable force to be at least 59 pN at a trapstiffness of 0.82 pN/nm.

Example 7. Ensuring Monomeric Rep-X Activity in Optical Tweezers Assay

We considered the possibility that the highly processive unwindingobserved in our optical tweezers assay was caused by multiple Rep-Xacting on the same DNA. If multimeric Rep-X had been required for highlyprocessive unwinding, then the majority of binding events (i.e.formation of a tether) would not have displayed unwinding activity,because single Rep-X binding is the statistically the most probableevent during the brief period of contact between the two beads. However,the majority of tethers formed displayed highly processive unwinding,suggesting that the processive unwinding is caused by a single Rep-Xprotein.

To further establish that the unwinding of the 6-kbp DNA was achieved bysingle Rep-X molecule, we repeated the experiment using beads incubatedin lower concentrations of Rep-X, thus decreasing the number of Rep-Xmolecules per bead. Consequently, Rep-X binding (tether formation) tooklonger and required more trials of bumping the two beads. As the Rep-Xconcentration was lowered. (20 nM, 4 nM and 0.4 nM) during thepre-incubation with 20 nM biotinylated antibody, the efficiency oftether formation was also reduced (7 out of 11, 9 out of 27 and 2 out of16 beads, respectively). However, the subsequent unwinding was still theprevalent behavior (7 out of 7, 8 out of 9 and 2 out of 2 tethers,respectively).

As another test to ensure that the highly processive unwinding was dueto a single Rep-X molecule, not multiple molecules, we compared theunwinding reaction of DNA with 75 nt vs. 10- and 15-nt 3′ overhangs.Since the footprint of Rep is reported to be 8-10 nt (S. Korolev, J.Hsieh, G. H. Gauss, T. M. Lohman, G. Waksman, Major domain swivelingrevealed by the crystal structures of complexes of E. coli Rep helicasebound to single-stranded DNA and ADP. Cell 90, 635-647 (1997)), 10 or15-nt overhang would increase the chance of single Rep-X binding. Rep-Xexhibited the same highly processive behavior on the short overhang DNAmolecules (17 out of 18 tethers formed with 10- and 15-nt overhang DNAvs. 21 out of 22 tethers formed with 75 nt overhang DNA, FIG. 3B, C),further indicating that the high processivity of unwinding is theproperty of a Rep-X monomer.

To test the possibility that the unwound ssDNA interacted withadditional Rep-X on the bead surface, possibly increasing theprocessivity of unwinding, we added 66 nM of E. coli ssDNA bindingprotein (SSB) in the unwinding reaction stream in order to render theunwound ssDNA inaccessible to other Rep-X molecules. Inclusion of SSBdid not change the highly processive behavior of unwinding (17 out of 18tethers formed in the absence of SSB vs. 21 out of 22 tethers formed inthe presence of SSB, FIG. 3B), suggesting that DNA unwinding by Rep-X ishighly processive whether the unwound ssDNA is sequestered by SSB ornot. This observation is probably due to the design of the dual opticaltweezers assay, in which the DNA is under tension only between the“front runner” Rep-X molecule and the streptavidin on the other bead.Binding of a second. Rep-X to the already unwound ssDNA should notaffect the measurements because the second Rep-X, which is also tetheredto the bead, cannot interact with the front runner that is tetheredelsewhere on the bead.

Example 8. Selection of Crosslinking Sites and Crosslinker Length

Open (inactive) and closed (active) form crystal structures of Rep andsimilar helicases were used as a visual guide. The target residue pairfor crosslinking and the crosslinker were selected based on thesecriteria.

One target residue of the target residue pair should be located on themobile 2B domain and the other target residue should be located on theimmobile body of the helicase (for example on 1B or 1A domains).Preferably, target residue pair should not be part of functionalhelicase motifs known in the literature to prevent detrimental effectsof amino acid engineering. Preferably the target residue pair should notbe conserved residues. Preferably the target residue pair should be asfar away as possible from the ssDNA binding sites. These measures reducethe potentially detrimental effects of the target residue mutations andcrosslinking on the basic translocation function of the helicase.

The target residues should be as close as possible to each other in theclosed (active) conformation of 2B domain, and at the same time shouldbe as far as possible from each other in the open (inactive)conformation. For example, the distance between the target residue pairshould be less than 15 Å in the closed form (measured from alpha carboncoordinates) and should increase by more than 30 Å during transition toopen form, so that a short crosslinker can prohibit the transition to aninactive (open) form. Residues that satisfy such criteria can bedetermined for helicases with known crystal structures in closed or openforms.

By sequence alignment, the corresponding crosslinking target residuescan be found in helicases with unknown structures to convert those tosuperhelicases, as well. Sequence homology models can also be employed.

Target residues should be preferably on the surface of the protein, andtheir side chains should be facing outward and more preferably facingtoward each other.

The crosslinker should be as short as possible, preferably only longenough to efficiently link the target residue pair in the desiredconformation. Crosslinker length should be considerably shorter than thedistance between the target residues in the unwanted conformation.

A representative 56 Rep homologs/orthologs with 90% identity to and 80%overlap are shown in Table 4, which are also shown in FIGS. 9A-G. Thetarget residues of FIGS. 9A-G were selected from one residue from domain1A or domain 1B, and one residue from domain 2B which satisfy the allthese considerations. For PcrA, or a homolog thereof, the targetresidues are selected from residues 92-116 of domain 1A or 178-196 ofdomain 1B, and 397-411, 431-444 or 526-540 of domain 2B. For Rep, or ahomolog thereof, the target residues are selected from 84-108 of domain1A or 169-187 of domain 1B, and 388-402, 422-435 or 519-536 of domain2B. For UvrD, or a homolog thereof, the target residues are selectedfrom residues 90-114 of domain 1A or 175-193 of domain 1B, and 393-407,427-440 or 523-540 of domain 2B.

TABLE 4 Rep homolog Organism REP_BUCAP Buchnera aphidicola subsp.Schizaphis graminum (strain Sg) REP_BUCAI Buchnera aphidicola subsp.Acyrthosiphon pisum (strain APS) (Acyrthosiphon pisum symbioticbacterium) REP_ECOLI Escherichia coli (strain K12) REP_HAEIN Haemophilusinfluenzae (strain ATCC 51907/ DSM 11121/KW20/Rd) REP_SALTY Salmonellatyphimurium (strain LT2/ SGSC1412/ATCC 700720) A0A077ZIR6_TRITRTrichuris trichiura (Whipworm) (Trichocephalus trichiurus) S3IEG5_9ENTRCedecea davisae DSM 4568 J1R585_9ENTR Kosakonia radicincitans DSM 16656K8ABZ8_9ENTR Cronobacter muytjensii 530 A0A060VJ91_KLEPN Klebsiellapneumoniae A0A090V5M6_ESCVU Escherichia vulneris NBRC 102420A0A083YZC2_CITAM Citrobacter amalonaticus A0A0J6D7T8_SALDE Salmonelladerby A0A085ITL8_RAOPL Raoultella planticola ATCC 33531 E7T4Q1_SHIBOShigella boydii ATCC 9905 A0A085GMM2_9ENTR Buttiauxella agrestis ATCC33320 A0A085HAK1_9ENTR Leclercia adecarboxylata ATCC 23216 = NBRC 102595D4BE16_9ENTR Citrobacter youngae ATCC 29220 A0A0H5PMJ7_SALSE Salmonellasenftenberg A0A0J1JQT3_CITFR Citrobacter freundii A0A0J8VI05_9ENTRCronobacter sp. DJ34 F5S3F4_9ENTR Enterobacter hormaechei ATCC 49162D2ZMA5_9ENTR Enterobacter cancerogenus ATCC 35316 A0A084ZTW9_9ENTRTrabulsiella guamensis ATCC 49490 A0A038CLT3_RAOOR Raoultellaornithinolytica (Klebsiella ornithinolytica) Q8Z385_SALTI Salmonellatyphi Q83IX8_SHIFL Shigella flexneri A0A0D5WYP4_9ENTR Klebsiellamichiganensis A0A0H3FM31_ENTAK Enterobacter aerogenes (strain ATCC13048/ DSM 30053/JCM 1235/KCTC 2190/ NBRC 13534/NCIMB 10102/NCTC 10006)(Aerobacter aerogenes) A0A0H2WUK6_SALPA Salmonella paratyphi A (strainATCC 9150/ SARB42) A0A0H3H1F3_KLEOK Klebsiella oxytoca (strain ATCC8724/DSM 4798/JCM 20051/NBRC 3318/NRRL B- 199/KCTC 1686) X7I146_CITFRCitrobacter freundii UCI 31 A0A0H3CTF5_ENTCC Enterobacter cloacae subsp.cloacae (strain ATCC 13047/DSM 30054/NBRC 13535/ NCDC 279-56)D2TH67_CITRI Citrobacter rodentium (strain ICC168) (Citrobacter freundiibiotype 4280) Q329V6_SHIDS Shigella dysenteriae serotype 1 (strainSd197) W6J7C4_9ENTR Kosakonia sacchari SP1 I2BE87_SHIBC Shimwelliablattae (strain ATCC 29907/ DSM 4481/JCM 1650/NBRC 105725/ CDC 9005-74)(Escherichia blattae) B5EZ38_SALA4 Salmonella agona (strain SL483)A0A0F5SGU2_CITAM Citrobacter amalonaticus G9YY11_9ENTR Yokenellaregensburgei ATCC 43003 A0A090UXU3_9ENTR Citrobacter werkmanii NBRC105721 A9MJ31_SALAR Salmonella arizonae (strain ATCC BAA-731/CDC346-86/RSK2980) Q3YVI6_SHISS Shigella sonnei (strain Ss046)D3RHB6_KLEVT Klebsiella variicola (strain At-22) Q57HT8_SALCH Salmonellacholeraesuis (strain SC-B67) B5RFS5_SALG2 Salmonella gallinarum (strain287/91/NCTC 13346) A0A089Q204_9ENTR Cedecea neteri A0A0H3BNR1_SALNSSalmonella newport (strain SL254) C9Y4T0_SICTZ Siccibacter turicensis(strain DSM 18703/ LMG 23827/z3032) (Cronobacter turicensis)B7LU77_ESCF3 Escherichia fergusonii (strain ATCC 35469/ DSM 13698/CDC0568-73) A0A0H3TAW8_SALEN Salmonella enteritidis G2S5J6_ENTALEnterobacter asburiae (strain LF7a) A0A0F7JC30_SALET Salmonella entericaI A7MQI4_CROS8 Cronobacter sakazakii (strain ATCC BAA- 894)(Enterobacter sakazakii) L0M8J0_ENTBF Enterobacteriaceae bacterium(strain FGI 57) A0A0K0HFU2_SALBC Salmonella bongori (strain ATCC 43975/DSM 13772/NCTC 12419) A8ACT1_CITK8 Citrobacter koseri (strain ATCCBAA-895/ CDC 4225-83/SGSC4696)

Use of shorter crosslinkers increase the efficiency of crosslinkingreaction by favoring the intramolecularly crosslinked species ratherthan intermolecularly crosslinked multimeric species. These rules alsoensure that the 2B domain is restricted to the active (closed)conformation, and cannot attain an open (inactive) conformation. Thusconformational control is achieved, and the possibility of 2B domain toswinging open to access an inactive (open) conformation is virtuallyeliminated.

Without being bound by theory, one possible explanation for the superactivation would be the decreased dissociation rate due to thecrosslinked protein encircling the ssDNA strand (indicated by thecrystal structure, so that the protein cannot dissociate from the ssDNAeasily. However, it was found that despite both Rep-X and Rep-Yencircling the ssDNA (as indicated by the crystal structure), only Rep-Xwas super-active. Thus, in order to create the super active helicase,immobilization of the correct conformational state of the 2B domain isnecessary.

Example 9. Identifying Suitable Crosslinking Sites in HomologousHelicases

Based on the crosslinking target site selection criteria established inExample 8, potential crosslinking target residues in helicases weredetermined using known crystal structures. By sequence alignment andstructural homology modeling, the corresponding crosslinking targetresidues are identified in helicases with unknown structures.Subsequently these helicases can be converted to superhelicase forms.For example, based on the criteria that the distance between the targetresidue pairs should be less than 15 Å in closed form and shouldincrease by more than 30 Å in open form, we identified the residues inRep, PcrA and UvrD helicases as shown in FIGS. 9A-G. Homologoushelicases are identified, for example, by 50% sequence identity and 80%overlap to the helicase with the known structure. For example, we found3147 such proteins homologous to E. coli Rep, 1747 proteins homologousto B. st PcrA, and 1209 proteins homologous to E. coli UvrD helicaseswere found (Tables 5-7, respectively). Then the correspondingcrosslinking residues are identified in any of the homologs. Forexample, we chose an example of 56 Rep homologs (Table 4), and found theregions where the crosslinking residues can be engineered (FIGS. 9A-G).Despite the fact that the three model superfamily 1 helicases, UvrD, Repand PcrA, have only 35-40% sequence identity, they exhibit >90%structural homology according to their crystal structures. Hence it isreasonable to expect a highly similar structural homology from theproteins with 50% identity to and 80% overlap to the helicase with theknown crystal structure; these are suitable candidates for crosslinkingin the superhelicase (−X) form.

E. coli UvrD (ecUvrD) has 33% sequence identity with E. coli Rep (ecRep)and 42% sequence identity with Bacillus stearothermophillus PcrA(bsPcrA). Highlighted regions in FIGS. 9A and 9G show the crosslinkingsites obtained from the open form and closed form crystal structures andthe criteria established in Example 8. These regions align well in thesequence showing that a sequence alignment can be used in helicases withunknown structures to determine the crosslinking target sites inhelicases with unknown structures. For example, the crosslinking regions(boxed sequences of FIG. 9G) in D. radiodurans UvrD (drUvrD) were foundby aligning its sequence to bsPcrA, ecRep and ecUvrD, 1A/1B residues:92-116, 182-200, 2B residues: 400-414, 434-447 and 528-544. drUvrD(Q9RTI9) has 33%, 36% and 41% sequence identity to bsPcrA, ecUvrD andecRep, respectively. These four proteins have 21% sequence identity as agroup. Only closed form crystal structures of drUvrD are known. Boxedregions shown in FIG. 9G are shown in the crystal structure of drUvrD(FIG. 11) to demonstrate the suitability of the regions forcrosslinking.

D. radiodurans UvrD (drUvrD, Q9RTI9_DEIRA) has only 1 Cys residue, and acrystal structure is known. drUvrD has 31 entries in the 50% identitycluster of the Uniprot database, some of which are mildly thermophilic(40° C.-68° C.; optimum growth at 60° C.), making them better candidatesfor helicase dependent nucleic acid amplifications. In certain exemplaryembodiments, a suitable UvrD helicase is selected from followingspecies: Deinococcus geothermalis, Meiothermus sp., Marinithermushydrothermalis, Marinithermus hydrothermalis, Oceanithermus profundus.Selected thermophilic ortholog species of drUvrD are shown in Table 8.

In another embodiment, the helicase is selected from those shown inTables 9 and Table 10.

TABLE 5 List of 3137 unique non-redundant helicases that have 50%sequence identity and 80% overlap with E. coli Rep. (Uniref50_P09980cluster, citable UniProtKB and UniParc accession numbers are shown).P09980 UPI00051877AD UPI00050997D4 A0A063KTD1 W0QF97 A0A0C3I5L6A0A0G3HMG0 UPI0002CB7C3E UPI00041BBC9F A0A0F9UW26 A3MZ01 UPI0005EDEB6EA0A069YUU2 UPI0002CB6CB4 Q31J65 UPI00057A0DA3 A0A0A7MFM3 Q4F7B3A0A069XK09 UPI0003EF7150 UPI0004A7511F UPI00042735E9 UPI00037127A9H2IB31 A0A090J554 A0A0B5IRM0 J3VUI0 A0A0B2JU34 W0QB54 UPI00039DFE76J2LMP0 UPI0005F8A649 B3PF82 A0Y242 D9P8T6 UPI0002D3CD90 A0A0E1CLG8UPI00036A6E72 W1J4L6 G7F259 B0BT27 UPI00031FC355 W8VIF4 E7T4Q1A0A068QMH5 F3BJI5 E0EII5 A0A034TPW5 A0A0J2JAV4 UPI0002C94EDFUPI000645DEF6 UPI0004641B09 A0A011P892 UPI0003B1A99F A0A0H4Z590UPI0004A19D90 A0A0J1C9T2 W1Z619 UPI00047C9D5E A0A090P8C9 A0A0H4YMK4A0A024KL85 A0A077P2A7 N6W1D7 W0QMN7 U3AJD4 A0A0H4ZZ54 A0A029K3Y0A0A077PGN3 G7EU46 B3H0C1 A0A0H0Y6H0 W1HB14 A0A029LST3 A0A077Q5V6UPI0003F6330E E0FKS8 D0WV97 A0A0H4ZHU7 A0A074IDU1 A0A0B6XFA1UPI0004175A32 E0E690 UPI00039CA46E A0A0G3T252 A0A070UMT6 D3VHW6 G7EBR9E8KIM6 K5UKZ2 A0A0H3GKB6 A0A0H0NZ89 N1NN52 UPI0002317DDE KOFXU5A0A061Q0T1 A0A0K0GSG9 UPI0004DACF34 A0A0A8NWA1 UPI000412695D A0A0D6UGN8D0X594 A0A0G8G0X7 UPI000543FDCF W1J8H9 A0A099L6T2 M9X512 UPI0006814400A0A060VJ91 A0A0H3MJI3 A0A077NN58 UPI0002DA5F7A D9P4Y2 UPI0006AA085CW1HUU8 UPI0002A2C4E1 A0A068RA85 UPI0003162143 UPI0002556C8CUPI00039C63E0 W1EG06 A0A0H0KMW7 A0A0A8LWM3 U1MBQ7 A0A0B0HCL6UPI0005EFDC1A W1HYL7 F4NQI1 A0A077PLN5 U1JK11 UPI00041A33FAUPI00063C4F58 W1EC12 A0A0G9FGL3 A0A077PEC1 U1IZP2 A0A081FYF7UPI0002B70576 V0AP50 UPI0005305422 D3V6L9 Q3IJF4 A0A0B3BWV0UPI00023755B4 W1E3B2 I2X0N8 A0A0J5FTN5 E6RJ61 A0A0B2DBM6 UPI00069FBA0AA0A080SZ33 UPI00063C108B A0A077N2X7 Z9K3Y4 UPI000337D905 A7K6B6 A6TGG4UPI00053AB8E4 UPI00037EA902 G7G5E8 D2TCP1 U4DZN2 W1BG54 UPI000390341BA0A094NXP4 UPI00034C744C UPI0001960924 L8XB50 Q57HT8 UPI0002C97E36F9QBX8 A0A0F4PWX2 V5Z3P5 UPI0005975B9F C0Q2V7 T8JFG3 UPI000364DEC7I3CJI7 UPI00065FAA51 UPI00039C5A41 A0A0G2NT58 A0A0J0IRL7 A3V094 A1T091UPI00054F6EBB UPI0005F0AEC8 V2QUW5 J1GHC8 UPI0002D3C2F3 UPI00036133E4UPI000554232A A7N1B5 B5RFS5 UPI0005EFAD7E F9SI11 A0A0A8UTG2UPI00044B0A82 U4K9S4 H3N5E4 A0A0D7LCG7 UPI00065FA69F UPI0002624F9FW0T2G5 U3ATU8 UPI00030AD7A3 UPI0005EFAEC1 UPI0003122A61 UPI000465C470A0A014LYL5 A0A0C1YTU5 A0A0J2K868 UPI00058EBC4A UPI00030ED944UPI0004E14814 E3DCZ6 A6AVT6 A0A0E2RBB6 A0A0J5E886 UPI0003151B6B A3YC72UPI00069D7BD3 UPI0003B1A0B5 A0A0G3S4X6 UPI0004D3B9EA UPI0002EE9ECEA0A0F4QZY4 D8MKR6 UPI0006A5B784 A0A0H3H1F3 UPI0005B485C4 F6CZU1UPI00036B36CA UPI00058F6E3E A0A0D0JDJ7 A0A068HB64 V2SM85 A6W1H3 B5JVS4I3IF96 UPI00069EC01E A0A0J8Z8W0 UPI0003AC5FE5 X7E960 M4U1U4UPI00066FB81F UPI0006A5FC2D UPI00066AC9B7 UPI00057BEBAB W1RWL7A0A0E4C1E5 UPI0005866DF0 UPI00026C4ED4 H3MDG6 A0A0C2ELY7 UPI00037950B0C4K8X2 UPI00037B74A6 UPI0005EF44E2 UPI0002F9F873 U9Y0B8 A0A0J8GYA3UPI0004762303 V5VG28 UPI0005EFA2F9 A0A0A0FJZ3 A0A0H0JVJ4 UPI0003F7784FUPI000558F88A D0C9R6 A0A0C1Z376 A0A077ZIR6 UPI0002CC3FC4 I1E2U9A0A0E9M3L1 A0A014AME8 A0A061PWI7 A0A0H7U3H0 A0A0D1Q3M4 Q0VLG2UPI000415F58C A0A062IS38 A0A090RFC6 A0A0H7UZL4 N2JD25 B4X088UPI0003736C3A A0A062RV86 F7S1D5 W1YGM8 N3FAJ0 A0A095S578 UPI00047DD116A0A014QUN9 UPI00037B6B31 W1F515 D7XDE9 U7FXU8 UPI00051B59BB A0A0J1AD48UPI0004DB80FB W1AVB8 J1R585 L0W9M5 A3WIV3 A0A011J490 UPI0002559C1CUPI00050B4286 A0A063XPH7 A0A095SJP8 UPI00058ED431 A0A009L4W9UPI0004226D1B UPI000699BCA0 UPI00046E61E1 UPI0003096010 A0A0B7IYA3A0A062ET27 UPI0005A04CC3 UPI0001BCF307 UPI0005CFB922 Q5QZ79 A0A0D7FE58A0A062B4C4 UPI00036751C4 UPI000291E920 F1ZPM5 A0A0B4XU81 N2J9F8 K6NBZ9UPI0004A71315 UPI0005CECD30 E9Z170 V4LHL4 H0JBY0 A0A062HCL9 G7LUC7UPI000699485E I2RDB4 UPI00048D495E UPI000307EC21 U1UEQ4 C6DHF7UPI0006995E89 G9YY11 UPI00066EC8FC UPI00056B6FFA N9HSF4 A0A0G4JUN2A0A0H7XVC5 UPI0005A8BE84 A0A0E0Y6R4 UPI000694928A A0A009GJZ6UPI0003AAB78C A0A0H7ZBU6 B7N271 F2G1X5 W9SXN9 A0A009HQM2 UPI0003A1DD75A0A0H8QAW7 A0A029IHD3 S5C3L8 G2E0G6 L9NZJ0 UPI0003AA3036 T9GN10A0A029HDM3 S5AI16 H5V6K1 A0A011IKX6 UPI00039E8574 U9YW53 UPI00005EFD38UPI00057CAFFD K8AD99 A0A062FYV6 UPI0003A8ECA5 E1ITJ2 UPI0005175495K0CYB3 UPI0002F4EC6B N8ZCZ6 UPI0003A98BB4 A0A029L915 UPI0004D67711K0EB21 K8AIR9 A0A022JJ04 UPI00039EFED1 S0UVF6 UPI00038F4A0A A0A0B3Y7T5UPI0002F0AB46 K1FLW5 UPI000532BCE2 I2SR57 UPI0004ED858A A0A075P1K0UPI0003747342 N8UCZ4 UPI0003AAAD9F I4S298 S1FND5 UPI000509BB4BA0A0D1MQD1 A0A010EBV1 U6ZJ94 D7ZUA2 S1LRE3 F5Z5Y6 F8FZI5 A0A009SCE5UP10005539481 A0A069X2C0 S1CH47 UPI0003556595 UPI0004706F0C N9GH50UPI00039F0A65 F3WQ14 L2VMG7 A0A010PKS2 UPI0004870A8C A0A0J1AU91UPI000399CBB8 E1J5T7 UPI0005EDE9CE A0A0C5ITU1 UPI0005B76FDE A0A009JME7UPI00039E8082 M9FM00 UPI0002CA0423 UPI0005D7D9DE UPI0004838E3BA0A062TQ97 UPI00039A51A2 S1HS46 UPI0004E384CD J2S9U8 A0A0C5RPA5A0A009QEV1 UPI00039E8236 D7YBV4 UPI000675EB35 UPI0002E4097E F0E436A0A010UBN4 V3TUV7 D7YG19 V0Z8Q3 UPI000641D99E J3E3S1 A0A062FMG3A0A0A8FH34 V0RR51 D7ZDM9 J3D7I1 T2HEW7 A0A013G992 UPI000532D788 V0AF21V5B2V3 J2T1B6 B0KQ68 A0A0J1A474 D2BYS8 E9YLU8 V1I8U1 J3EAI0UPI00048196DC N8YU24 C6CGH3 I6CD40 T9SAP6 A0A0J6H1C3 A0A084C8X6 K6LHM0A0A089V9A1 E1HWT5 A0A0I1J330 J2MEX6 A0A099E6T7 A0A062DHL7 A0A0A3ZID8A0A070FCA9 A0A0J3J6C1 UPI0004653130 A0A088NVW6 A0A009TH79 Q9K599 V0TJ44L0M8J0 UPI000370037D A0A0F7Y5R7 A0A0J0ZSY5 A0A0B2TLM6 S1H2A1UPI0002C957E6 UPI00054B503C A0A099N7M2 F5I0P2 E0SKR5 E6BNJ7UPI00033C37F9 UPI0005A8A319 V7DDK2 A0A010K6Q8 J8TIB8 D6I334UPI0003EF8387 L1M697 L0FS45 F5IED5 UPI00050455DF S1ESS4 UPI00040AF69DA0A0F4VBH8 V9V750 N9KF69 A0A0B3XKC6 S1CGG8 W8XM69 J2XVS9 A0A059V3S3S3TL87 D0KC43 I2WCN5 F0JWU2 J2NB92 B1J4P9 A0A009MSS6 A0A0H3IEB9A0A070DIV3 A0A0J2D6H5 S6JBD8 UPI000627A5A1 A0A0J0ZPS4 A0A0J5XQ16A0A079D8A1 L5GW15 J2UYW0 UPI0003A45A0C A0A010GGU7 UPI000505D45BA0A080I8Z6 S1P4F9 I4JZZ1 UP10004901875 A0A0J0ZKR3 A0A0J6AFU6 L4IUS8UPI0006187646 A0A0J8G475 UPI000312CC5F B0V8Y9 A0A0B3Y0I6 L3IPS4UPI0002CA3627 A0A038GJS8 V6JD09 A0A0E1PVY4 UPI0004E7AD0D V6FPL2 S1IH44J2NTP4 A0A0C1IE71 A0A0E0WM10 UPI0005010995 F4T625 UPI0001FB5104UPI00054BC40B 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UPI0002677FD2 C9P8Z5 UPI00037D0EF9A0A078LYT9 A0A0F4NNJ4 I1XRY3 D4BE16 F2JTM7 UPI00037114DA A0A099RR43E8LUB9 UPI0006831C68 UPI0005794143 A4C7W3 B5FCU9 A0A0D9ATR1UPI0002E710F1 G4AZ10 UPI000260B9F8 UPI00039F0D67 Q5E1T0 I4JQR3UPI00031D8F80 L8UHA2 UPI00028307CF UPI0002558A91 B6EP51 I7A9J8 F7YIE8UPI00067CF184 UPI0006660AB8 UPI0005B86EE4 UPI000247865A K5XG83A0A066UM26 UPI0006815E34 A0A0E0VC55 A0A098G8R1 A0A090IP02 A0A0A1GK07F9S363 H0KC96 T8Z104 UPI000326FBA0 T2L6T4 UPI00040F147B UPI0003102D21A0A0E1YSI3 S0YAD1 UPI00037E68F8 L9UDC2 UPI0004194356 A0A099LPD8 X2JQA6S0X3V2 UPI00034D7EBF UPI00037E36DB UPI0003B39C7B UPI0003167DE1UPI00067FF093 S1DN83 UPI00056CF143 A0A0D7UZT0 UPI0005BA4855UPI0002E0F676 C6AQX1 S0XCC9 A0A0B8V8Y9 A0A0F9VK34 UPI0005B7EB79A0A0C2P7J1 UPI0006A71057 S1E4M9 U4TCI2 A0A0D5LWG2 A0A0C1EK43 A0A0C2JL07G4ABR6 T9IPB3 A0A0H4R6J4 G4F9U9 U1AEW3 UPI00031E1DE0 L8UIY4 T6LBU7A0A0B8USV8 A0A0B1PVT1 UPI000617AEB5 A0A0A5I590 Q7MYL0 S1GJ06UPI000368EE93 H0J1C6 UPI0006182BB7 UPI0003043227 A0A022PH42 D8ADY5A0A095VW14 UPI00048842D1 Q7NQR9 U3BS43 W3VA31 S0VUC6 A0A0C5UZZ5UPI0002D5FF35 A0A0J6LGT4 UPI000571B2B4 A0A0A0CQ83 B7LU77 W8FU49UPI000556BD4F A0A0D8ZDY8 A0A086WW56 UPI0006203273 A0A070K818 M5DYB2A0A0C3I966 UPI0004907BDE F9T770 A0A0F7LMM1 A0A062XSU2 UPI00046D03A5UPI0004843630 C5BIA4 UPI0005F11AED UPI00055C23C6 UPT0002C9B880 R4YVB5F7SNI3 UPI0003800078 UPI000699D72A A0A0J9EYL2 UPI000651920D H2G1G7UPI0004AB49AE UPI000382D783 UPI0005F118D8 C7BQK5 A0A0F3TFS9UPI000379F9FB A0A060B1U5 UPI0004227F3C A5L7N9 U7R5K0 A0A0E2U8U7UPI0001EC45E8 A0A0D6EF56 UPI00035C7304 UPI0001F55149 A0A081RWC7 C8TYS8C6XCN5 G9EBD3 UPI000369273A U0FTU6 UPI00058BF6A9 A0A0J2E1P9UPI00035FCCBC A0A0F4RA35 W8KPW4 UPI00030715AD T0PH03 UPI0005B2C8D4UPI00058D9CB0 W1N5Q2 D5C0J9 UPI0006303856 A0A085JM95 UPI0002C95CB8A0A0A0BIG5 UPI0004CE4C17 A0A0A3AKX9 E3BPB4 A0A095VZP6 A4WG32UPI0002DE5ECC A0A081K8A4 UPI0006A9F0F0 E8MCD7 UPI00046F3C99UPI0003420E0F F5T1S3 UPI000477FE94 F7NR22 Q7MQG8 UPI0004A3375FUPI00036F08F6 A0A0F9NIL3 A0A094JA28 A0A066T3V5 A0A087IWU5 A0A0F9VYU9UPI0005C4EB5F C0N7C2 A0A090KED5 A0A080LJV3 UPI0006A98AC0 I1XM63UPI0002C91779 F6DAI6 A6FE65 X2GZR9 UPI0006A98232 I2JF40 A0A078L9V2UPI0005C9F562 UPI00030A12FE A0A0J5P3I3 UPI0002482DB8 F9ZY05UPI000512B6F1 W0DYM2 UPI0002DF92C0 A0A0F2P6J6 F9REI7 A0A0F5V836UPI0002CC209C UPI00022C089B UPI000464C875 W7R0N4 UPI0004F5E7D9 S6GDK2A0A0D7LJA0 UPI0006844205 UPI000427CAB0 UPI0004752339 UPI00031835E8S6HCZ4 A0A073VC48 W7QF72 B9CXM2 UPI0004E147DD UPI00031E4412 I8U5K5X7I146 UPI00058DEE31 C5RZQ1 UPI000479BC17 A0A0H2MLA0 H3ZE84 X7HFY3UPI000289826B UPI00035CAB1A UPI00047B5018 UPI0006195856 J1YGP6A0A089Q204 U7NY76 UPI0003B481E4 W9V341 UPI00067F4562 H2IWS7UPI000675D9DD A0A098RE99 UPI0004212DE4 UPI0005C15FEC A0A097QPF1UPI0005D339A3 UPI0004D8E29C E1VCA4 A0A0C4WTU2 E0FET2 A0A0H0Y092 I0QQI6B7UMN3 UPI00030B6E67 C1DJY5 UPI000248B5E1 UPI0002F588B8 UPI00038060C2H4I3H7 UPI0006148CBA M9YDT7 J4TTN5 A0A0G9M026 H8NUJ7 H4JUI7UPI0005B789BB UPI0004E1F9C3 S9YCL8 UPI0002DD07EC A0A0H3FLE0 H4KPD2S2KK42 W0E158 I2NC81 A0A0B4IM65 UPI000554A929 H4L565 UPI0003674641A0A0F7K0Q6 UPI00031355EC A0A0A3EMP0 A0A085G3A6 H3KW73 UPI000343B180UPI00048BF236 E0F2E9 K5V6E0 UPI00041ABFF4 H4LJK6 UPI000376C869UPI000395D43A E0F8J5 UPI00066B2D3D UPI0003089400 H4IZH1 S5T4K5UPI00046F29D9 E0EW49 UPI0005F9A9ED UPI0004719479 H4JFJ8 K0C8L7A0A0F5ARC9 E2P8X6 M7RIV0 UPI00058F6D4E H4K9V6 A0A0F9YVG2 Z5XTJ6 W0Q1J8K5TSH9 C8NBT1 E3XW38 UPI0004057986 UPI0002AA68F1 UPI0005856421 C9QC04UPI000660E94E H4IIJ0 D2TWS6 A0A0F4S821 A0A0B5BWF7 A8T649

TABLE 6 List of Bacillus stearothermophilus PcrA homologs that have 50%identity to and 80% overlap. 1747 members of Uniref 50% identity clusteris shown (citable UniProtKB and UniParc accession numbers are shown).P56255 J7M5U5 T0TN09 A0A0I6PI88 R3VBE4 UPI0005CD7F53 S7T032 A0A0H2UUM0F8LQ03 A5LVX9 E0G4K8 UPI000417C0DE UPI00051815BF Q1JLF2 C2LSM3UPI0005E41D7E E6GJJ0 UPI0005CD905E A0A098L684 UPI0003C7B0E5UPI00065FC663 S7YIM5 S4DY07 G7SM20 U2YC97 UPI000254D55F UPI00066E20BDUPI00066CDBC6 C2H162 UPI0004062509 G8N340 Q1J6A6 E3CPD8 E1LG87 R3D1M0UPI0004051F87 T0Q4M4 A0A0G2V0F7 W3XXV6 A0A0I6BPW7 X6SFW2 UPI000411FB5CA0A063Z1I8 M4YYG1 T0T6T2 A0A0I9JBK7 X6RK63 UPI0004022BA7 L7ZT56A0A0G4DFH5 UPI0002AEC4C7 UPI0005E02B0B R4CW85 UPI0004188987 V6VMU8UPI0001E10349 UPI00065FB970 UPI0005DC8263 X6RKD4 UPI0005CE22CD Q5L3C0UPI00000D9968 UPI0003167399 UPI0005E61B75 X6SVN5 UPI000400A66BUPI0005CD09ED A0A0G3U9S6 A0A0A1DXP2 UPI00066DCA04 R4DBG2 UPI0005CD5B4AA0A0D8BW89 Q48T98 A0A0F3HAQ8 E0Q0Y1 C7WG78 UPI0005BE8F33 A0A087LEV1UPI00038E29D2 V6Q5R6 UPI00066CD043 C7W6F8 UPI0005CC9805 UPI00066FD17EA0A0H3BYK1 A0A0C2HKT0 UPI00066D16C5 R3EDY6 UPI0005CC91DA UPI000519CC89UPI00050BF55F UPI0004E153F7 UPI0005E1DA5A E0GYA3 G5L3H4 A0A0G3XVN0UPI0004BE2C5F UPI000288F7E6 UPI0005E04403 R3L6W4 UPI00040C9E90 A4IJY5UPI0004F92D8A A0A031IBW4 A0A0I6R2B8 E6FGS3 UPI0005CE3BAA UPI0005CCA9FFUPI00066C9AA9 B1YJ16 UPI0005E64F07 UPI00031E170D UPI0005CCD039 S5YVH0E7PYJ1 UPI0006AA2516 UPI00066B4226 E2Z449 UPI0005CD5201 UPI0004DF596FUPI0004BE34CB UPI00047948D2 A0A0I7U0N9 UPI0002F39C67 UPI00040A255CA0A0E0T7W1 UPI0004BE2973 UPI000683717C UPI0005DBFF52 E6IN81UPI000419AA26 UPI0006A962ED C5WGR7 UPI00041CB696 A0A081PQV0UPI00031A4BB4 UPI00040A186C UPI00017E56F4 F5U8K1 K0A8A5 E8KBG6 S4G3W1UPI0004221FBB UPI0001D581E8 A0A0E4B7C5 U6BA96 UPI00017C1A3F S4FMR9UPI0005CDC9EE UPI000424F449 Q5XBW2 UPI00047AE0FA A0A0I8Y7H2 C7VZ55UPI0004624A9B A0A0J0V9H4 Q1JGI8 UPI000494D4D3 UPI0005E6A918 C7UKX9F4EF32 A0A093UDD5 UPI00044FEC83 UPI0004792A31 A0A0F2E3V6 A0A0E1C082UPI0005CF3160 UPI000539F1EA K4Q9V2 UPI000494A958 UPI00066DA31C R1KTX5UPI000400B1DD N4W917 UPI000617EC21 U7USF2 UPI0002AF45D8 UPI00045B8E48UPI00040B8112 UPI00055386C0 UPI0003C7BD0F A0A0E0UWM6 UPI00066EE4B6R2UDI9 B9WTD9 UPI0005590F34 A0A0F5P2U0 A0A0E1R8Q5 A0A0I8ZZI4 R3B3J0UPI00041EA41C UPI00020D9901 UPI0003C7D5B0 A0A0E1Y218 E0SZL7 R3J9A8UPI0005CEFECB I8UBH1 UPI0006181569 A0A0B8RF49 UPI00025ABE1BUPI00032DCFB5 UPI00041FE6F0 UPI000555B2C6 I7WIP9 UPI000035D23AA0A0I7UZI3 UPI00032F5E7D UPI0005CE9E1B UPI00059000A4 A0A0G2V4A4UPI0001B43587 UPI0002313C8F A0A0H1TNE2 UPI0005CED2C1 A0A089XIR5 I1ZL68UPI0003591B75 UPI00066E2824 R3C367 UPI0004128A2C T0TNV3 E8K3X2UPI0005128D3B A0A0I8XVX0 R3VC46 UPI0005CF2FFD A0A0H1RMM4 W1Y2G0UPI0003EC8641 UPI0005E6A956 V7ZS55 UPI0003FA464B D2BQM2 W1VGX2A0A0H4NBP6 X8KE98 C7YF48 UPI0005CDDD5E A0A0A7T646 UPI0002F353ECA0A097B674 UPI00027EA587 R3W0C9 UPI0005CD2C43 T0W2G0 F8DFS6 A0A0F5Z989A0A0E7WHF5 C7UTX1 UPI0005CE703E H5SYV7 UPI00031E513E E3ZRG0UPI0005E9B66A C7UDZ4 UPI0005B9BF22 A0A0B8QL14 E7SC56 UPI0006282029A0A0I5V7V4 C7WW04 UPI0005CCB173 G6FEQ4 UPI00021BD63E UPI00052F1B16UPI0005EA0304 R3KHA0 UPI0005CD2248 Q9CGH6 I2NTT3 A0A0H0TBR3UPI00066ED988 R3KQT9 UPI0003F9DC72 F2HJH7 UPI00065F8C03 UPI0001975CA4A0A0B7LAG3 U7S5K0 A0A075SIP2 T0V8Q3 UPI00066A6FE6 UPI00003CA336 M5K5E4R3Y2U6 UPI0005CCB671 U6EMQ9 U5P378 A0A0H3GCW7 M5K8F4 R3ATF7 D5AH63A0A084A9A3 A0A0F3H3Y1 A0A0H3IUG2 M3IAM8 R1LJ35 UPI0002195DB2 Q02Z69W1VGW0 UPI000431635A D3H992 R3NBA7 UPI000367B16E U5PKA6 UPI00066BB1D3UPI000396C49F UPI0005DADCC0 R3PMA1 UPI00040780E6 A2RL58 U5PAT9UPI00057E5CD1 A0A081QTN0 C7WTA5 UPI00047DE9FC T2F5M7 UPI0004E25F53UPI000541B044 A0A0F2DL31 C7V916 A0A0D6A4Q8 K7VSC5 UPI0006600922UPI00065E0E60 E9FH57 C7VLS1 UPI0003079325 T0VAM5 UPI00066E3612UPI00059B4903 K0ZJQ5 R2VJF4 W1SU53 UPI0006172C2A V8BFU1 UPI00064CDA40UPI0005EA108D C7VRD8 K6DTK1 G8P2F6 UPI0006605290 A0AJL2 A0A0I8KXT3R3I2U9 UPI00058D7294 S6FGR2 UPI00066EBF4B UPI0004F3EE62 G6R9C9 R3I1C1UPI0005F025AA UPI0005838B59 F9M2F0 UPI000570B4C0 A0A0I8MDZ1 U7SAR7UPI00055EBF2F T0UFT5 UPI00066C8E1F H1G7W7 X8HMR1 R3DJQ9 A0A0F3RNZ7Y1QH73 UPI00066A3914 UPI0004D59A43 J4Q880 R1JZF5 UPI0005534922UPI000629DD6D UPI00066CB902 G2ZBE3 W3XTS5 R4ALC3 E7FSU1 Y1Q968 E3CEE7A0A0G2WHH3 UPI00066C1FD6 R4B5K9 G2SLV9 UPI000376C4D7 UPI00066DB632A0A0G2W8X5 UPI00066BE89C Q837V7 A0A0G8G5N5 A0A0D6DXQ0 UPI00066D30C4Q92AP9 UPI0005E8CD89 A0A0E1RBS1 R6S5W5 R2SEZ2 K8MRD3 Q8Y6C9 K1ABC3A0A0F5AWZ1 A0A0G8G9A4 UPI0003F7ADC6 UPI00066D94ED A0A0G2VQM3 K1AGK6A0A059N1T2 A0A0B4B1V1 F9DT42 UPI00066CDADB A0A0G2VT50 K0ZZ87 S7U796F7R373 UPI00054EA513 A0A0F3HXT7 UPI0001EBB745 A0A0I6M9Y8 D4EPU4UPT00062CB1C4 UPI00030811AC I7IYF1 UPI00066D78F0 UPI0005E36939 U6RZD7UPI000409F2F6 UPI00036C3403 UPI000345D943 E6J2G2 UPI0005E225FD E6ISD3C0WZU5 S0KSL1 Q8ES83 UPI0002FD7F0C A0A0I7LKZ4 J6NDX6 A0A0G9GFB1A0A0A5GAU1 R2R7H3 W1TUK9 A0A0F2DPD4 S4GCT9 V4X193 UPI000414CEF0 R2VFE1I0S6A8 UPI0005E11B6C C0X2D5 A0A0F4HGR5 A0A078MEE0 R2RQ91 UPI000660E090A0A081QG94 J6PVS6 U2HDV7 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UPI00031AFBDD UPI0005E22D3B A0A0J5WFP0 U5UIJ5UPI00058E169D UPI000319EA31 E8KV65 E1LS08 A0A0J5YA29 UPI00042996E4UPI000624F4AA UPI0002FFFF03 UPI0002E8600B A0A024DEK7 UPI0006A9C586UPI00042A2929 A0A0A8JEM1 Q8DTY6 F8HD36 UPI0005E2SBC5 C8NHG1 A4VUA8UPI00047BFF10 UPI0002B59757 A0A0E2QHQ8 UPI0005E30B11 UPI0005874702A0A0H3MVK6 I9B3V6 UPI00035CDC0C F8LX97 E1M4S7 UPI00066C1DCEUPI0005CD2519 A0A075K9S A0A084GLL3 A0A0F6BVJ6 UPI0005E14CCC D4YVQ1UPI000409C6E9 I9NQ12 A0A084H1D9 A0A0E2RHF6 F5VXC9 E6FS51 UPI0003FE3351UPI0004883363 E6TWN0 UPI000264F340 A0A0E8T7V0 UPI0002EA5AD2UPI00041E695A S4NRZ4 K1LG40 UPI0000E563DC E6KMR2 S4CP69 UPI0005CDA05AJ9W320 F2F7J1 UPI000660EC4F UPI0005E76F14 UPI0003FECF16 UPI0004038E95F4FSH6 J1GP52 UPI00066C13CA UPI0005E0C70E F2MQT5 UPI0005CE89C0UPI000403AE07 F8HYK0 F8LIZ1 UPI0005E6F0D4 UPI0002A3D37C UPI0004018E0CA0A084HBI0 UPI00044D3C3A G2GTJ2 S7YYN6 E0H8L5 UPI000404D8AB D3FTF3A0A0C6G2S0 X8J9A0 A0A0I8TLZ0 R1W0H5 UPI00041CFDD8 U6SL82 U2W3N6UPI00066AA528 E9FJW6 S4FW64 UPI0005CDED2A UPI00036426F2 A0A0E1ENC5A0A074IU47 UPI0005E93C3F R3UP49 UPI0005D236D1 UPI00047A28D8 Q99ZE1

TABLE 7 List of E. coli PcrA homologs that have 50% identity to and 80%overlap. 1029 members of Uniref 50% identity cluster is shown (citableUniProtKB and UniParc accession numbers are shown). P03018 K8BG21UPI0002C8F355 UPI0005A9630D UPI0003EF5338 A0A0J0DJ77 A0A0G3HMD3A0A060VDV3 V1HN20 A0A0A3YR40 UPI0001F6648A A0A0J0SUX3 U9ZBE3 A0A0E1CLV1UPI0002C9B17D UPI0005EB7A8B UPI000678B341 A0A0J0M6S9 A0A071CB77 W8V249UPI0006811593 UPI00058E54A4 A0A0K0IDG2 A0A0F0XZS7 S1J559 A0A0J2G3Q6A0A0J4VXC9 H1C573 UPI0002CC80BD UPI0005D0A9E9 V2S4E7 A0A0H4Z3E1UPI00025C7C5C UPI0005CD86D3 A0A0J5K2Q0 A0A0C8UHF8 A0A073G662 V0AU35UPI0005304A96 UPI00044E7286 A0A0H3MJV2 A0A0C9HTD3 I2SQY0 A0A0H4YPU3UPI0002CAB12A UPI00037EE7F7 A0A0E0VDJ7 Q8Z3B0 B3X3W4 A0A0H5AHT5UPI0005CCA08F A0A0J0GVC5 A0A0G2SID2 A0A0E7LC59 N2GY76 W1HG62UPI000330B244 A0A0H0CXK2 UPI000542989F W6J799 W1F3C2 A0A0H4ZLF1UPI0002CCBAB8 V3PV69 A0A070RYI3 UPI0004DA823D E1HNQ6 W9BQA0 F3WPX7A0A0D1KFS4 H3MUW4 V8MJC9 A0A070SNS2 A0A0H3GGJ9 A0A0F6YD20 H5V6H2A0A070H7E9 UPI00049F5927 H4URJ5 A0A0K0GRR7 K8DQF9 D7YBR7 UPI0003BC8E89N3EUQ7 M9G7C2 A6TGJ6 K8C9V5 UPI0002C8B609 UPI0003910486 UPI0004693D87N2IIQ0 A0A0G8G1B7 F5VR52 UPI00063CD924 A0A090UJD6 UPI0002CCC2BC S1HRC3UPI00058FD925 A0A0D1QDQ1 A0A0C2AR33 UPI0004977D3D F5N8N5 D8E9M5UPI00058F49FC A7MQJ8 M9I6S8 UPI0003EF3FD7 UPI0006A5855E A0A074HPP5W1HTQ0 V5U5I0 N3K330 UPI0002CC54C7 Q83IW7 L2VEY2 W0ZY91 UPI0005187950I6CD07 A0A0G3S4T9 A0A0C7MG10 K5CJK9 F4T661 K8D2A7 E7SHD7 A0A0H3HA95A0A0G3KPN2 D7YG58 F4V8D3 UPI0002CA0405 B2TUW9 A0A0E0WSN3 Q0SZ04 W1BJG9F4TMH3 A0A0J0I5H8 UPI000390185B A0A068H452 D2ABY6 N2QEY3 A0A029LAE5UPI000579149A UPI0005EEDAF9 UPI0004A0FDEC A0A0F6MJ85 A0A069YVJ3 U9YHH0UPI0006650689 B5RFP5 A0A0H0GX62 F5P1J3 A0A070Y0G0 A0A080IB93 N2J8A4UPI0004733206 UPI0002CB804F A0A0F6EK00 A0A073GWJ7 A0A083YZ93 A0A063XKV2UPI00026721AF UPI0002CB6B71 I6BAB7 V0RR87 UPI0005C48DC6 UPI0005C63608UPI0003A80309 UPI0004D7856B UPI00050B7641 V0ACC7 UPI0005A8BF01 E7T4T6A8ACW1 A0A084ZTZ9 UPI00050B2FF7 N2RS67 UPI0003710649 A0A0G2XIC2A0A0A5IRH8 A0A062Y212 I6FW66 A0A069XHA8 A4WG04 I6DJM1 A0A0F1WNC5A0A064DKM1 UPI00067F497D A0A079H1K8 A0A0J8F6L5 K0WUD4 V3DAP7 A0A080EWZ9UPI000530716C A0A074IWT6 UPI000666003A E7TCS8 A0A0E2K1D2 UPI000668F9A7A0A0B1RCP6 F8XAY4 A0A0I1EMQ9 Q31UH5 UPI0004D8D514 V5AU63 B6I4F4A0A074HJR7 A0A0J5U9E7 A0A085HAH3 UPI0003EF42B5 UPI0002CC06C9 E9TMV0V1BCC5 A0A0J6MG09 A0A0J5L085 UPI0005A87CA8 A0A0B1FRQ9 UPI0004D72F99A0A080HWB3 UPI0006684F9F I2BE57 UPI00016A0FB4 S1FP27 UPI00025ABCDFI2X3X5 UPI00058D9C39 A0A0F1BI78 UPI000496CFDD S1L396 UPI000627F480A0A070FA84 A0A0A5RML6 A0A0J0RXX3 UPI0006ABEED8 S1CI55 UPI000326F8B9L3K8J5 A0A085ITJ0 Y1GM95 UPI0004646130 L2VN93 A0A0F4HLT5 A0A080GHX3A0A038CQJ1 E8C7D9 B7MR33 A0A089U9W2 T9FRL3 A0A073FPS6 A7ZU18 V2JXK2A0A029IIQ6 D2TV17 H5E8S0 S1GRU8 UPI0002CA1DFD V1LV18 A0A029HFI5UPI000667BF5F I4S2D3 H5J8D4 A0A0F3LUY4 E8D343 A0A0J9KSZ0 UPI0006207A91V8FG33 D8ERJ1 UPI0002481DE4 S4INC0 A0A0H0KN67 I2X271 I2RVR0 D6I369A0A0J8LYC2 E7ZSL5 UPI0002CB91AD A0A0D6IZH2 A0A070D8G2 A0A071CFC4A0A0J8MSQ3 A0A038D0Z4 C3SKC2 B7MH77 A0A026UZE9 M8SKZ8 A0A0J8HX73 E7YUD9A0A0H8C28 A0A0E2KYP4 A0A028CBA2 S1EV38 A0A0J8QFU8 S4J0L5 V0VC55UPI000512AED4 V0U5F8 S1CHB8 A0A0J8KFX8 E7YT71 V0SS57 UPI0002C8F6BFH5A4E5 I6CYG1 A0A0J8IWX2 E8F002 T8ZCA9 G9YXY2 G2AN47 H5IRF1 A0A0J8NMQ9E8EDF3 N4MZW5 UPI0002CC829C K3QIJ3 I2WCK0 A0A0J8JFF5 G5LW37 A0A070K8G3UPI0004B001CB A0A070SY69 A0A071DAV1 A0A0J8M4K3 E8FVB6 W1BBJ0UPI0004E37056 I2UC63 A0A070DJ71 A0A0J8HJM3 E7VG83 V0XWV9 UPI0002C98364M9EF05 A0A079D807 A0A0J8M7Y3 E8AMN0 L2X7H9 UPI000267F8CE A0A027TGT7V0YB46 A0A0J8LY21 V1PEK5 T8JFJ4 UPI0002673104 A0A0E1SZY6 D8AZQ6A0A0J8K6V3 E7ZUE1 T5TRC8 UPI00066D844D A0A0E2U398 L3IME4 A0A0J8JD30E8B3Y5 H4I3L1 UPI0005083EE7 A0A027ZJG3 I4J587 A0A0J8KYA0 G5PV87A0A0J3V9C5 UPI0002CABCFF C8TL04 T9CEL0 A0A0J8M2A9 T2Q2W7 N4NRN6UPI0002C94803 A0A028E3K3 A0A070ULP7 A0A0J8KZU2 E7VUY1 U9Z163UPI0005309A93 A0A026HN93 H4V876 J1GHE8 E8BI66 X7NZ16 A0A0J5MIB1A0A025G7T3 F3VD13 UPI000472C058 S4IB83 S0YT63 UPI0002CC9136 K4VZX0K3KG98 UPI0005F8A7CD E7XYR0 H4JUM1 UPI000269547E K4XMA4 G0F7H1UPI0005ED3E27 V7WD74 A0A073H2N3 UPI00034730CE A0A0H3XBG3 E6B0S3 S1I248A0A0J6D7Q1 A0A017I312 UPI0003910F49 H9UZ11 E0J3Y2 UPI000512EA8D V1U5Z5A0A080ECD1 UPI00057C0D33 C8UJJ5 A0A037Y8I6 UPI0002CB816A E8EQ65 L5GW49A0A0G3PID9 A0A0A8UGD6 A0A0E2U8R4 UPI0006815C5F E8GKX8 S1P4I2 A0A0J4WXG0UPI0005B345AD E8Y8R2 A0A0H7LQT5 E7Y7G4 V2T0S1 V3D6C7 I6FW96 A0A0E0U5P0UPI0002CA127F E7WDV6 A0A073UI66 A0A060UYE6 UPI0004713F51 B7L973UPI0003BB4FC5 E8H1P2 V6FB56 M7P8V6 UPI0002CC83F4 E3PP00 UPI0002C92D2DS4M012 J7RN24 W8XG71 A0A0F5SGW9 A0A0E0Y7I2 A0A0F0YW97 E8CHG5 S0XLH4V3KJ79 UPI00069BE650 A0A0E3H4E0 A0A0F6K2Y9 S4JDI0 A0A064T2Q3 W8XNG3A0A069X2G5 C8TYP3 UPI0002515E81 V2N400 M9F528 A0A098GXV9 A0A080FIP4A0A0E1M3W0 UPI000699EF6E S4LF58 S1D3C3 UPI0004D54D12 A0A073T7U4A0A090L9E8 V3IA60 E8DQ33 H4KPG7 UPI0005EDDA48 A0A0F1AYX8 A0A0A0F8P2A0A0I2HXR0 V2P0K5 V8KDE4 UPI0005F08B1C A0A0J1YCS9 UPI0005E69EA7UPI000579D3C9 A0A0H5PMN6 A0A070P4C7 UPI0002CC5FF6 A0A0J0HLB6 W1G679A0A0J9AH48 V2ISV7 U9Y365 R0D8R6 A0A0F0RX59 C0VZH1 T9ARP5 V1SA88 V0YN02UPI0002CB96B1 UPI0005CAF560 UPI0006978729 U9ZZ52 T2PQM4 V4B7K3A0A0H0HV04 A0A0D7LBX3 UPI0003EE8CC5 B7NFB5 E8AAS2 E9YLR4 S3IGV1 H4JFN3W1WHJ6 UPI000445D59E E8CTA8 M8LCT6 UPI00068E1050 A0A0J1M123 D8ASL4UPI0003EF87D0 E7WWF9 M9GJI0 A0A0J4LFX4 X7HIN0 UPI0006695A36 A0A0F4BA88E8FMH0 L3Q9J9 A0A0H0CH29 A0A064CY91 UPI0002C95A23 V5KL37 E7WRB0 S0X3S7A0A0J0K9Y5 S0TTK4 W1XFI9 V2MBS7 E7X696 V0Y7G3 W7NZ36 UPI0006520C97UPI00050ADC02 A0A0H3T6D2 E8GBI9 A0A070PK74 B5EZS8 UPI0002CC3250 W1WI72X5GT01 V1K5C8 H4L599 H7EDN3 UPI0002CB3E81 UPI0005097CC3 UPI00056EBE4BX0NNF5 S1EDJ9 UPI0002E3BEE2 UPI0004DA7107 Q8KI59 J1QP03 V2AKC0 T9IU72UPI0004E2422C Q05311 UPI00044FBFBE A0A0J2C8A9 E7VT49 A0A079Y2R2A0A0J5MX43 A0A0D6IPI8 UPI0005CCFA15 E1ITF3 A0A0J6JML4 A0A0G3J263A0A085HQF9 A0A0E8MI42 Q9R2U0 A0A0D1CQK0 V1I8L5 H3KWA8 A0A078LAH7A0A021WR03 UPI00050A604C V6FP78 E8E0U7 H4LJP0 A0A0H0R1B4 S5IH33 V0V674V2ASN4 S4JVA6 T6GSY1 UPI0005575061 V2KFI5 A0A0A7A0U6 UPI000627EB24E7ZFI6 T6LNG9 UPI0004D8B75C A0A0H3SHZ2 E2X518 UPI000237C903 V2H9R2H4IZK5 UPI000452C3C5 E8XJD9 Q329Y9 A0A0J5L635 E8BMX2 T5NEX8 R8WLR8A0A0H3NUG9 A0A0J1JGH9 A0A085GMJ6 G5NKF6 N3MX37 S0XDX3 V7QPA0 W1FYY2UPI0005E94CC5 V2A9V9 A0A029P4R5 A0A0A1B385 V1H945 A0A0A1R5N6 U1VBA4A0A0G2MMZ1 A0A027YRP2 UPI00016C8460 A0A0F6B9B3 A0A073VBC0 UPI00066656EEV1MAM1 L3PWK5 UPI000675DF85 A0A0F7JES7 I6FY95 A0A0H3FP62 B3YFM1 S1GVU2A0A0E1LGB9 UPI0005F937F6 A0A0D7LIV8 UPI0005014921 A0A0H2WUN6 M9K6A8D2ZMD4 L0MA89 X7I032 UPI00063C446F V1XNT7 T9TBM0 UPI0003ED146BUPI0004B98CEC D4BE43 UPI0002CAC6D5 A0A0H3S2Q8 D7ZK11 A0A0E2A5Z6 E1I441UPI0001C3403D UPI00026947D6 X2KCL1 L3NT10 A0A0I2G829 D8ADU8 G5P1B6UPI0002B60DFC A0A0H3IIW8 H4K9Z1 UPI0002C8DC1E UPI0001FB4B2C G5LGM2I6FIC1 C0Q3C2 S0VUG2 UPI00066659C9 D7XDB2 A0A0H2VE91 A0A073VVJ1 V2NKZ3M2P544 UPI0003EF3546 UPI00050B0CB8 Q1R4C1 UPI0003F93F50 A0A0H4VNJ1E3XW04 UPI000370A2F0 A0A0I0YDW9 W8ZQE8 L4IV51 V7UEH9 S0V315 A0A0J0PQF7UPI0003FF3A54 A0A024KJK2 A0A0J8YSU2 M4LQ08 N2JTA5 E6WHH6 UPI00067E3DB0UPI00050B495A UPI0002CAC228 Q57HQ6 H4IIM4 UPI00057BE5A7 UPI00050ABBE7A0A090ND62 A0A0F0R0L1 A0A089GCQ8 A0A026RVL8 UPI0003BF7FA1 UPI00050BC0F6A0A024L7U7 U2MK71 S5HQI6 E9XUJ2 T8XXA5 UPI0001FB4D65 I0VX51UPI000575034C A0A0H3BQS9 A0A017JGC0 A0A0H0BBN2 A0A0I2EFX3 C9XT80UPI000282E630 V1SQB1 D6JHC8 A0A0F3XJB2 V6E727 UPI0003027365UPI0005307602 A0A0H3RDJ9 T5ZU25 A0A085PA08 V0VKJ6 K8BR96 UPI0006A629C6V0GAX0 Q8X8P5 D6IG48 UPI000589632A W0AUM0 UPI0006A6039B A0A0F0IT73B1LLY4 L4UZM9 UPI0002A4D3B7 UPI0002B9DE03 UPI0002CC68E2 A0A0D5WNL4R6TVJ8 L3C1J2 UPI000628182D K8A0N1 UPI0002CCA014 A0A0F2ZMT8 A0A0G3JMG2UPI0004D7F7DF UPI00062757E2 UPI0005196C1F A0A0D5WY30 A0A0G2NZ21 D3QXA5UPI0006800C6C UPI00053A6F37 E3G3X3 A0A0K0HFZ6 V5ZRD0 D3H4V1UPI0002CCBDA0 UPI0005CEF8A7 A0A0B5INH2 UPI00056ED442 V7IJT2 A0A023Z641UPI0002CB0C5E A0A0H7L7Z4 UPI0006969E0D UPI0002CA6A43 V2D935 C6EG01A0A0J1LKQ0 A0A0I1QVM4 A0A0J8ZBK9 F1ZPQ8 B5Q5I2 B7UND0 A0A0G2NT28UPI0004643C70 UPI0002EE2722 E9Z1A2 V1RGT6 L9HYA4 V2PRV4 A0A0I2RPB4A0A0J1RJH0 I2REU8 X4BR52 A0A0H3PUC7 UPI0002CBC0D7 UPI000281D683A0A066P4B2 UPI0005EA4E43 UPI0004A8DEFA A0A0F6GUU7 B3HAV2 A0A0I0V6U1F5S3C5 UPI0003BCDF55 G5P176 Q3YVF3 UPI00050A9E00 W1WLH4 A0A0E2M6M6UPI0002C935D9 G9WCL2 A0A0F6FES0 UPI0004D4FB82 UPI00069A9A9D A0A0J0P9D3A0A090V7I4 G5MAR0 A0A0G3KBA3 M8PMP0 A0A0H7RCS5 A0A0J0VSA8 A0A089Q428UPI00067C89D7 A0A0F6CBC7 UPI000483DDB5 UPI00050B3740 A0A0J0QVP3UPI00039807B5 UPI00067AC747 J2YWY3 A0A0D7ESI8 W1ASV1 A0A0J0LCW8UPI0004DA8D8C UPI00069F6BC0 B7LU43 A0A0J0DP92 W1DW14 A0A0A6EFN1UPI0004635F02 UPI0002A6DF22 A0A025C616 UPI000352C78C J2X0N7 A0A0F1A8N0UPI000463708C UPI00067D0E8D A0A0H4S4M4 G4C8R9 G5LGM3 A0A0F1HGJ1 R9VNE2UPI00028DE27E A0A0H2Z4N7 UPI0005AA8C72 UPI0002B9DB1F A0A074TPI3UPI0002CC3EDA UPI0005F857AB F0JWA1 UPI0002CC9A6F X3YLW0 A0A0J9AGF8UPI0002695288 UPI0005797D3A UPI0005EAF698 UPI0005C674E6 UPI0004381BCDUPI000668E496 UPI00034D611A J5W6W9 D7ZU66 UPI0006658EEE UPI0002AEB5B0A0A0J1SRM4 M7RF80 A0A0F1L5B3 UPI000696EBE1 A0A0J4TS24 G5QS93 A0A0H0ABS0K8AAR4 UPI0006675A7B UPI0006995D61 A0A0C7L099 G5MAU7 A0A0J2H3P7A0A0I2D6J9 E1J5X4 UPI00053B46E6 Z5CP12 X3UNX0 A0A0E2R9B6 M8KEA1 E6BNN4UPI000681EBB3 D3RH84 B3PGX1 UPI0004B58C5B UPI000574FBCF D7XMB5UPI0002C925C5 B5XYK3 UPI0002DB7E81 A0A0J2FBS7 UPI0002A1343F A0A079F6E9B1ERG0 UPI0005CC1957 UPI0003B61D19 A0A0J9AI33 UPI000537C7CA A0A071AVK4UPI0002CB7FF2 UPI000666ABBF UPI00037AE6F5 A0A0J8Z8W7 A0A0J8Y5W3A0A079FJR3 A0A029K3W3 R5WI88 UPI00040A8AC5 H3MDK3 UPI000472771CUPI0005AB1B13 A0A029LTL0 A0A089PHR7 A1SQW8 A0A0G3PTS1 UPI0002B580C6UPI0002CBB03B UPI000390DC2A A0A0H3CV27 G5QNH0 A0A0I1EU55 E8DEL4UPI00069C71E8 S5N2R7 A0A0H0C242 G5S3E4 UPI000669A104 E7XIB6 X5MS66G2S5G8 A0A0F0TB45 G5SJ34 W0BDW4 UPI0003915F4D UPI000614634C A0A0F2AUK2A0A071M1C1 X3XE62 G8LKV0 UPI00038FA10B UPI0005ED8E6D A0A0J0JZA2UPI00035E9F50 UPI000689139D A0A0J0TK85 A0A0F6TXR6 V1GX81 UPI0006769073F4W269 G5R9I0 A0A0J0GZG0 UPI00037F6D42 A0A0G3QEA8 A0A090U681UPI00038FA53E G5P3F2 A0A0G4BNQ9 UPI0006145584 UPI000666A5AA A0A023V4X1A0A0B7GI73 UPI0003D2FA70 A0A0I0T9Z3 UPI0004DAE8E7 UPI000315529EA0A0I2BUS0 A0A0G2MHY8 A0A084CN62 R4Y7F0 A0A0J8XHN7 A9MJ02 R8WJE0A0A0H0DHS6 UPI00068E1512 C8T0H7 UPI0003BECD47 S1HNI3 A0A0J0IRI6A0A0J0SSF2 UPI0005D093A3 F4VLD8 H3N5H9 UPI0003BB87D8 A0A0F3YGX7A0A0J0B472 A0A0H4R3L7 F4SRL8 K6KT52 UPI000353E7DB UPI0002CB932DA0A0J0ENJ8 A0A0B8UZ32 F4NQE6 A0A0H3ENI0 A0A0F5B4P9 S0UJP4 A0A0H0DM28A0A0B8V3X1 K8B2N0 UPI0004D4C5A1 UPI000250C01F M8X9A7 A0A0J0PB20 U4TEK6UPI0003A800E6 UPI000598DBB2 N4NWV1

TABLE 8 D. radiodurans UvrD and its Orthologs in Thermophilic SpeciesProtein Accession # Entry name names Organism Gene name Q9RTI9Q9RTI9_DEIRA DNA Deinococcus radiodurans (strain DR_1775 helicase ATCC13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/ NCIMB 9279/R1/VKM B-1422)FORMJ1 FORMJ1_DEIPM DNA Deinococcus proteolyticus (strain Deipr_0885helicase ATCC 35074/DSM 20540/JCM 6276/ NBRC 101906/NCIMB 13154/ VKMAc-1939/CCM 2703/MRP) H8GTP8 H8GTP8_DEIGI DNA Deinococcus gobiensis(strain DSM uvrD2, helicase 21396/JCM 16679/CGMCC 1.7299/ DGo_CA1449I-0) C1CVA3 C1CVA3_DEIDV DNA Deinococcus deserti (strain VCD115/ uvrD,helicase DSM 17065/LMG 22923) Deide_12100 A0A016QL30 A0A016QL30_9DEIODNA Deinococcus phoenicis DEIPH_ctg079orf0093 helicase Q1J014Q1J014_DEIGD DNA Deinococcus geothermalis (strain Dgeo_0868 helicase DSM11300) D3PR99 D3PR99_MEIRD DNA Meiothermus ruber (strain ATCC K649_05745helicase 35948/DSM 1279/VKM B-1258/ 21) (Thermus ruber) A0A0D0N7B7A0A0D0N7B7_MEIRU DNA Meiothermus ruber SY28_04645 helicase E8U932E8U932_DEIML DNA Deinococcus maricopensis (strain Deima_1926 helicaseDSM 21211/LMG 22137/NRRL B- 23946/LB-34) D7BGJ6 D7BGJ6_MEISD DNAMeiothermus silvanus (strain ATCC Mesil_1937 helicase 700542/DSM9946/VI-R2) (Thermus silvanus) A0A0A7KLI4 A0A0A7KLI4_9DEIO DNADeinococcus swuensis QR90_10300 helicase F2NK78 F2NK78_MARHT DNAMarinithermus hydrothermalis Marky_1312 helicase (strain DSM 14884/JCM11576/T1) A0A0F7JIM6 A0A0F7JIM6_9DEIO DNA ‘Deinococcus soli’ Cha et al.2014 SY84_01165 helicase E4U8J8 E4U8J8_OCEP5 DNA Oceanithermus profundus(strain Ocepr_1221 helicase DSM 14977/NBRC 100410/VKM B- 2274/506)L0A7L7 L0A7L7_DEIPD DNA Deinococcus peraridilitoris (strain Deipe_3622helicase DSM 19664/LMG 22246/CIP 109416/KR-200)

TABLE 9 36 seed sequences of UvrD-like helicase group PF00580 ADDA_BACSUEX5B_MYCTU O53348_MYCTU PCRA_GEOSE Q9ZJE1_HELPJ UVRD_ECOLI ADDA_LACLMHMI1_YEAST O66983_AQUAE PCRA_MYCTU REP_BUCAP UVRD_HAEIN EX5B_BORBUO24736_THETH O83140_TREPA PCRA_STAA8 REP_ECOLI UVRD_MYCGE EX5B_CHLTRO25569_HELPY O83991_TREPA Q46538_DICNO REP_HAEIN UVRD_MYCPN EX5B_ECOLIO26611_METTH O84614_CHLTR Q9Z7D4_CHLPN SRS2_SCHPO UVRD_RICPR EX5B_HAEINO51319_BORBU P73465_SYNY3 Q9ZCJ7_RICPR SRS2_YEAST Y340_MYCPN

TABLE 10 Selected Low-Cysteine or No-Cysteine Wild-Type PcrA HelicasesPcrA with no cysteine from L. citreum MK20 /gene=″pcrA″MSVETLTNGMNNKQAEAVQTTEGPLLIMAGAGSGKTR /locus_tag=″LCK_00476″VLTHRIAHLVQDLNVFPWRILAITFTNKAAREMRERIAA /EC_number=″3.6.1.-″LLSEDVARDIWVSTFHALAVRILRRDGEAIGLAKNFTIID /note=″COG0210L;TSAQRTLMKRVINDLNLDTNQYDPRTILGMISNAKNDM TIGR01073″LRPRDYAKAADNAFQETVAEVYTAYQAELKRSQSVDF /codon_start=1DDLIMLTIDLFQSAPEVLARYQQQFEYLHVDEYQDTND /transl_table=11AQYTIVNLLAQRSKNLAVVDGAGQSIYGWRGANMNNI /product=″ATP-dependentLNFEKDYPNAHTVMLEQNYRSTQNILDAANAVINHNNE DNA helicase PcrA″RVPKKLWTENGKGDQITYYRAQTEHDEANFILSNIQQLR /protein_id=″ACA82309.1″ETKHMAYSDFAVLYRTNAQSRNIEESLVKANMPYSMV /dh_xref=″GI:169803691″GGHKFYERKEILDIMAYMSLITNPDDNAAFERVVNEPKR (SEQ ID NO: 53)KFLTFAEMMHNLRQQSEFLNVTELTELVMTQSGYRQMLAEKNDPDSQARLENLEEFLSVTKEFDDKYQPEDPESIDPVTDFLGTTALMSDLDDFEEGDGAVTLMTLHAAKGLEFPVVFLIGLEEGIFPLSRAMMDEDLLEEERRLAYVGITRAMKKLFLTNAFSRLLYGRTQANEPSRFIAEISPELLETAYSGLSRDKTQKKTLPFDRKMQRATATTYQATPVTKITNGVTGGDQTSWSTGDKVSHKKWGVGTVISVSGRADDQELKVA FPSEGVKQLLAAFAPIQKVDSelected Low Cysteine count thermophilic PcrA helicases >tr|B5Y6N2|B5Y6N2_COPPDMALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDL DNA helicaseSKLNEAQKQAVTAPPKPLAIIAGPGSGKTRVLTYRALFA OS = CoprothermobacterVKEWHLPPERILAITFTNKAADELKERLGRLIPEDGRIFAproteolyticus (strain ATCC 35245/ATMHSFAARMLRYFAPYAGISQNFVIYDDDDSKGLIEDI DSM 5265/BT) GN = pcrALKQMNMDTKRFRPNDVLNHISAAKARMFDCNTFPEFIR PE = 4 SV = 1 (SEQ ID NO: 54)QRYGSWGYYFDTVHQVFMTYERLKEQSQALDFDDLIMVLAQRMEDRPELREMIAGLFDLVMVDEFQDTNFAQYQMLLYMTNPHYSGMNNVTIVGDPDQSIYGFRAAEYYNIKFRIDDYNPEVVFLDLNYRSNRTIVSDASALINDSPSALFERKLESIKGAGNKLILRRPFDDADAAITAAFEVQRLHKMGIPYEEIAVLMRTRALTARVEREFATRNIQYHIIGGVFPFFARREIKDILAYLRLSRNAMDRVSLKRILTMKKRGFGTASLEKLFNFAEENKLTLLEAMKAAVESLLFKKLSMNDYLESLYTLIQTIQEIAEPSQAIYLVMEQENLLDHFRSISKSEEEYIERTENVKQLISIAEESADMDDFLQRSALGTRENNGGVEGVAISTVHGVKGLEFQAVILYYVTDGFFPHSLSVTTAEKEEERRLLYVAMTRAKEHLIFYVPYKQPWGNGFEQMARPSPFLRSIPKELWDGKPNEIESLYAPYSPQQKWSE >tr|E8MZN5|E8MZN5_ANATUMDSLEHLNPQQRAAVTASAGPVLVLAGPGSGKTRVLTF DNA helicase OS = AnaerolineaRIGYLLSQLGVAPHHILAVTFTNKAAREMQSRVEKLLGH thermophila (strain DSM 14523/SLQGMWLGTFHAICARILRREQQYLPLDANFVIFDEDDQ JCM 11388/NBRC 100420/QALIKRALRDLNLDEKLYRPTSVHAAISNAKNNLILPED UNI-1) GN = pcrA PE = 4 SV = 1YPTATYRDEVVARVYKRYQELLVSSNAVDFDDLLLYA (SEQ ID NO: 55)WKLLNEFSTVREQYARRFEHILVDEFQDTNLAQYELVKLLASYHRNLFVVGDEDQSIYRWRGADYRNVLRFEEDFPDRQKILLEQNYRSTQRVLDAAQAVINRNRNRTPKRLKSTPEHGEGEKLVLYEAVDDYGEAAFVVDTIQQLVAGGKARPGDFAIMYRTNAQSRLLEEAFLRAGVPYRLVGAMRFYGRREVKDMIAYLRLVQNPADEASLGRVINVPPRGIGDKSQLALQMEAQRTGRSAGLILMELGREGKDSPHWQALGRNASLLADFGSLLGEWHRLKDEISLPSLFQRILNDLAYREYIDDNTEEGQSRWENVQELLRIAYEEKGLTAFLENLALVSDQDTLPENVEAPTLLTLHAAKGLEFPIVFITGLDDGLIPHNRSLDDPEAMAEERRLFYVGLTRAKKRVYLVRAAQRSTYGSFQDSIPSRFLKDIPADLIQQDGRGRRMGRSWQSESRRSWDDNYAGTWGSRPERAKPSHAPILQPRFKPGMRVKHPSWGEGLVVDSRIQDEDETVDIFFDSVGFKRVIASIANL EILS >tr|E8PM35|E8PM35_THESSMQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGA DNA helicase GSGKTRTVVHRVAYLIAKRGVFPSEILAVTFTNKAAEEM OS = Thermusscotoductus RERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKP (strain ATCC 700910/GFVVYDEDDQTALIKEVLKELGLAARPGPLKALLDRAK SA-01) GN = pcrA1 PE = 4 SV = 1NRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALKA (SEQ ID NO: 56)QGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEARFVAEEILRLGPPFDRVAVLYRTNAQSRLLEQTLASRGVPARVVGGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALRVAAEVLPRPALRHFLALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPEDYEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSAPGEARASKPGAYRGGEKVIHPRFGQGTVVAAMGDEVTVHFEGVGLKRLSLKYADLRPVG >tr|E8PL08|E8PL08_THESSMLNPEQEAVANHFTGPALVIAGPGSGKTRTVVHRIARLI DNA helicaseRKGVDPETVTAVTFTKKAAGEMRERLVHLVGEETATK OS = Thermusscotoductus VFTATFHSLAYHVLKDTGTVRVLPAEQARKLIGEILEDL (strain ATCC 700910/QAPKKLTAKVAQGAFSRVKNSGGGRRELIALYTDFSPYI SA-01) GN = pcrA2 PE = 4 SV = 1ERAQDAYEAYKEEKRLLDFDDLLHQAVHELSTDIDLQA (SEQ ID NO: 57)RWGHRARFLIVDEYQDTNLVQFNLLRLLLTPEENLMAVGDPNQAIYAWRGADFRLILEFKKHFPNATVYKLHTNYRSHNGIVTAAKKVITHNTQREDLDLKALRNGDLPTLVQAQSREDEALAVAEVVKRHLDQGTPPEEIAILLRSLAYSRPIEATLRRYRIPYTIVGGLSFWNRKEVQLYLHLLQAASGNPESTVEVLASLVPGMGPKKARKALETGKYPKEAEEALQLLQDLRAYTGETGEHLASAVQNTLHRHRKTLWPYLLELADGIEEAAWDRWANLEEAVSTLFAFAHHTPEGDLDTYLADILLQEEDPEDSGDGVKIMTLHASKGLEFAVVLLPFLVEGAFPSWRSAQNPATLEEERRLFYVGLTRAKEHAYLSYHLVGERGATSPSRFARETPANLIHYNPTIGYQGKETDTLSK LAELF

Example 10. Cysteine Reactive Crosslinkers and Alternative Crosslinkers

Bis-maleimide crosslinkers with contour length varying from 6 to 25Angstrom were used as exemplary crosslinkers (Table 2): BMPEG2, BMOE,BMH, DTME, (1,2-Phenylene-bis-maleimide), and (SuccinylBis[(phenylimino)-2,1-ethanediyl]bis(3-maleimidopropanamide)).Alternatively bis-maleimide crosslinkers such as BMPEG3, BMB, BMDB,(1,4-Phenylene-bis-maleimide), (Bis-maleimidomethyl), and(N,N-[Dithiobis[(carbonylphenylimido)-2,1-ethanediyl]]bis(3-maleimidopropanamide))or homobifunctional vinylsulfone crosslinker such as HBVS can be used.An alternative crosslinker can be of any crosslinker of desired lengththat fits the criteria set forth in Example 8 with suitable functionalend groups. For crosslinking two cysteines, suitable end groups can beany of the maleimide, haloacetyl, iodoacetyl, pyridyl disulfide,vinylsulfone and other suitable moieties. Table 11 shows examples ofbis-maleimide linkers with corresponding lengths.

TABLE 11 Selected Bismaleimide Crosslinkers Spacer Arm CompositionCrosslinker Spacer Arm Length (Å) (between maleimide groups) BMOE 8.0Alkane BMDB 10.2 Cis-diol (periodate cleavable) BMB 10.9 Alkane BMH 13.0Alkane DTME 13.3 Disulfide (reducing agent cleavable) BM(PEG)2 14.7Polyethylene glycol (PEG) BM(PEG)3 17.8 Polyethylene glycol (PEG)

Example 11. Alternative Crosslinking Methods to Cysteine Crosslinking

As an alternative to cysteine crosslinking chemistry, one can introducea pair of unnatural amino acids for crosslinking with linkers usingdifferent chemistries as defined herein. This may be advantageous overcysteine engineering, because it may eliminate the extra steps of sitedirected mutagenesis of potentially interfering native cysteines andpotentially detrimental effects of such mutations in other relatedhelicases. For example, it was shown herein that in the PcrA helicase,there are two native cysteines that are highly conserved across diversespecies (FIGS. 4A and 4B). The mutating out of these two cysteines inPcrA from Bacillus stearothermophilus reduced the ATPase activity bymore than 80%. However replacing all five native cysteines in Rep fromE. coli had a very minimal effect.

Alternatively, a target residue pair can be introduced, one of which isan unnatural amino acid and the other is a cysteine. Alternatively, onecan introduce two or more pairs of target residues, preferably each paircan be specifically targeted with specific crosslinkers that employorthogonal chemistries so that unwanted inter-pair crosslinking isavoided (for example, one pair of cysteines and one pair of unnaturalamino acid residues) for enhanced conformational stability and activity.

Example 12. Unnatural Amino Acids as an Alternative to CysteineCrosslinking

There are nearly one hundred unnatural amino acids (Uaa) that have beengenetically incorporated into recombinant or endogenous proteins. TheseUaa provide a wide spectrum of side chains that can be covalentlycrosslinked using a homo or hetero bi-functional linker with suitableend groups. Additionally a multi-branched multi- or homo-functionalcrosslinkers can be used for secondary conjugation other chemicals,biomolecules such as a DNA polymerase enzyme, in addition to the maincrosslinking reaction. Uaa can incorporate specific reactive groups tothe specific sites on the proteins, such as aryl iodides, boronic acids,alkynes, azides, or others, or they can be post-transcriptionally orchemically modified to prepare for desired crosslinking chemistry.Examples of Uaa include, but are not limited to, homopropargylglycine,homoallylglycine, azido-phenylalanine, azidohomoalanine and others. Uaamodification and crosslinking reactions include, but are not limited to,azides and cyclooctynes in copper-free click chemistry, nitrones andcyclooctynes, oxime/hydrazone formation from aldehydes and ketones,tetrazine ligation, isonitrile based click reaction, quaricyclaneligations, copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition,copper acetylide to activate terminal alkynes toward reaction withazides, Staudinger ligation, cyclooctyne reactions, and Huisgencycloaddition. Suitable end groups of these crosslinkers would include,but are not limited to, azide, alkyne, succinimide, phosphine, etc.

Example 13. Selected Super-Family 1B (SF1B) and Super-Family 2 (SF2)Helicases

Selected SF1B and SF2 helicases are described herein. In an embodiment,the helicase is RecD2. In an embodiment, the RecD2 helicase is from D.radiodurans. Selected target residue pairs for crosslinking and thespecific distances between the pairs, in RecD2 are shown in FIG. 12 andTable 12.

TABLE 12 Selected Crosslinking Pairs for 5′ to 3′ SF1B SuperhelicaseRecD2 Backbone C—C RecD2 distance in Å ALA632 ILE170 18.0 ALA632 ASN17117.0 PHE635 GLY200 18.0 1B domain amino acid 2B domain amino acidBackbone C—C (RecD2; D. radiodurans) (RecD2; D. radiodurans) distance inÅ ARG 410 (B-sheet) ASN 596 (loop) 12.91 PRO 413 (B-sheet) PHE 603(loop) 13.04 GLN 414 (B-sheet) ASN 596 (loop) 11.13 GLY 415 (loop) GLU601 (loop) 8.38 PHE 416 (loop) ARG 417 (loop) 6.36 ARG 417 (loop) ASN599 (loop) 12.43 GLY 418 TYR 598 (loop) 11.00 LEU 411 (B-sheet) PHE 603(loop) 13.62 ARG 417 (loop) ARG 417 (loop) 10.14

RecQ helicase has a winged helix domain (denoted by WH, shown in greenin FIG. 13 and FIG. 14) that rotates 90 degrees and makes contact withthe duplex in the unwinding conformation (Mathei et al., “Structuralmechanisms of DNA binding and unwinding in bacterial RecQ helicases”Proc Natl Acad Sci USA. 2015 Apr. 7; 112(14):4292-7). In an embodiment,stabilization of the WH domain of RecQ leads to superhelicaseactivation. Stabilization of the closed form of the WH domain can beachieved by crosslinking it to the catalytic core using the residuepairs shown in Table 13.

TABLE 13 Selected Crosslinking Pairs for Superhelicase RecQ Backbone C—CCatalytic domain WH domain distance in Å PHE221 VAL470 7.91 GLU219ARG514 5.61 LYS212 GLU467 8.90 PHE221 GLU467 6.52

RecQ1 helicase also has a winged helix domain (denoted by WH, shown ingreen in FIG. 15) that rotates 90 degrees and makes contact with theduplex in the unwinding conformation. In an embodiment, stabilization ofthe WH domain of RecQ1 leads to superhelicase activation. Stabilizationof the closed form of the WH domain can be achieved by crosslinking itto the catalytic core using the residue pairs shown in Table 14.

TABLE 14 Selected Crosslinking Pairs for Superhelicase RecQ1 Zinc fingeralpha WH beta hairpin Backbone C—C helix domain amino acid domain aminoacid distance in Å MET429 TYR564 12.17 VAL431 THR566 8.31 MET429 ALA5658.77 MET429 THR566 7.10

5′-3′ SF1 superhelicase T4 Dda (FIG. 16) is known to unwind dsDNA as amonomer, and has sequence similarity to E. coli recD (exonuclease V). Inan embodiment, stabilization of the tower/hook and pin domains leads tosuperhelicase activation. Stabilization of the closed form of thetower/hook and pin domains can be achieved by crosslinking them usingthe residue pairs shown in Table 15. Wild-type T4 Dda has 439 aminoacids, a 5′-3′ unwinding polarity, and 5 cysteines. It is a DNA helicasethat stimulates DNA replication and recombination reactions in vitro,and has been suggested to play a role in the initiation of T4 DNAreplication in vivo. It acts by dissociating and associating with theDNA molecule being unwound, interacting with UvsX and binding tightly tothe gene 32 protein. Selected crosslinking pairs that parallel SF1Ahelicases are located in the tower/hook and the pin domains based on thecrystal structure (FIG. 16) and are listed in Table 15.

TABLE 15 Selected Crosslinking Pairs for Superhelicase T4 Dda 1B domain(pin) 2B domain (tower/hook) Backbone C—C amino acid amino acid distancein Å THR 91 (B-sheet) TRP 374 (Alpha helix) 9.77 TYR 92 (B-sheet) TYR363 (Alpha helix) 11.78 TYR 92 (B-sheet) TYR 363 (Alpha helix) 11.73 TYR92 (B-sheet) LYS 364 (Alpha helix) 10.42 GLU 93 (loop) LYS 364 (Alphahelix) 6.83 GLU 93 (loop) ALA 372 (loop) 9.25 GLU 93 (loop) PRO 373(loop) 10.45 GLU 93 (loop) SER 375 (Alpha helix) 10.38 GLU 94 (loop) TRP374 (Alpha helix) 8.25 GLU 94 (loop) ALA 372 (Alpha helix) 8.25 GLU 94(loop) SER 375 (Alpha helix) 10.73 GLU 94 (loop) TRP 378 (Alpha helix)8.58 VAL 96 (B-sheet) LYS 381 (Alpha helix) 12.55 VAL 96 (B-sheet) TRP374 (Alpha helix) 12.36 VAL 96 (B-sheet) TRP 378 (Alpha helix) 10.56

Structural data have been Obtained for the SF1B RNA helicase Upf1 (5′-3′SF1B RNA/DNA helicase) in complexes with phosphate, ADP and thenon-hydrolysable ATP analogue, ADPNP (Cheng et al, 2006), although astructure with bound RNA remains lacking. These structures reveal aconformational change that accompanies binding of ATP and which is verysimilar to that which occurs during catalysis in SF1A helicases such asPcrA.

Example 14. Identifying Suitable Crosslinking Sites for Immobilizing 2BDomain at a Particular Rotational Conformation Between the Open andClosed Form

It has been shown herein that the closed and open forms captured in thecrystal structures are the active and the inactive states of the Rephelicase, respectively, which can be interconverted by a 133 degreerotation of the 2B domain around an axis. Therefore, the activeconformation can be defined through definition of the range of arotational angle, θ (theta), relative to the closed form with θ=0 (FIG.17). For example, in an embodiment, Rep-X becomes a superhelicase ifθ<40 degrees. In addition, arresting the helicase in an intermediateconformation, such as, e.g. θ=40 degrees, may allow a new function.While immobilizing the 2B domain at an angle θ=40 degrees, it was foundthat residue pairs distances increase more than 10 Å when θ changes from40 degrees to 0 degrees (to closed form), and increase more than 20 Åwhen θ changes from 40 degrees to 130 degrees (to open form). Positionsof residues at the desired θ, can be interpolated from open and closedform crystal structures via rigid body rotation of the 2B domain aroundan axis. Having performed this calculation for θ=40 degrees of Rephelicase, it was found that 2B residues that satisfy this criteria areresidues 515 and 518-525, and the residues on the rest of the proteinstructure satisfying the criteria are residues 543-547. For example,crosslinking residues 521 to residue 547 on with a crosslinker with alength of about 10 Å, restricts the 2B domain to a conformation of θ=40degrees. Similar to restricting the 2B conformation to θ=0 degrees(closed form), corresponding residues to restrict in helicases withunknown structures can be determined via sequence alignment.

Rigid body rotation of the 2B domain around a chosen axis can convertthe closed form to the open form or vice versa. In the case of E. coliRep, the chosen axis intersects the alpha carbons of residue ILE371 andresidue SER280 or residue ALA603. In an embodiment, the chosen axisintersects the alpha carbons of residue ILE371 and residue SER280. Thetais the angle of rotation around this chosen axis from the closed formtoward the open form. According to this definition, theta is 0 degreesfor the closed form. In the case of E. coli Rep, theta increases to 133degrees when it is rotated around the chosen axis to obtain the openform. Theta for the open form may vary between different helicases.

Thus, in an embodiment of a modified helicase described herein, thefirst amino acid and second amino acid, together with an axis vectordefined by an alpha carbon of ILE371, from which the vector originates,and an alpha carbon of SER280 or an alpha carbon of ALA603 of E. coliRep helicase, define an angle, theta, wherein theta is about 355 degreesto about 25 degrees in an active conformation. In an embodiment, thetais about 355 degrees, about 0 degrees, about 5 degrees, about 10degrees, about 15 degrees, about 20 degrees or about 25 degrees, or anyincrement or point between about 355 degrees to about 25 degrees. Inanother embodiment, theta is about 0 degrees in an active conformation.In an embodiment, theta is about 60 degrees to about 155 degrees in aninactive conformation. In an embodiment, theta is about 60 degrees,about 65 degrees, about 70 degrees, about 75 degrees, about 80 degrees,about 85 degrees, about 90 degrees, about 95 degrees, about 100 degrees,about 105 degrees, about 110 degrees, about 115 degrees, about 120degrees, about 125 degrees, about 130 degrees, about 133 degrees, about135 degrees, about 140 degrees, about 145 degrees, about 150 degrees, orabout 155 degrees, or any increment or point between about 60 degrees toabout 155 degrees. In another embodiment, theta is about 133 degrees inan inactive conformation. In an embodiment, the axis vector is definedby an alpha carbon of ILE371 and an alpha carbon of SER280 of E. coliRep helicase. In another embodiment, the axis vector is defined by analpha carbon of ILE 371 and an alpha carbon of SER280 of E. coli Rephelicase.

Example 15. Examples of Thermophilic Orthologs/Homologs of UvrD, Rep andPcrA

Based on the crosslinking target site selection criteria established inExample 8, and analogous to identification of suitable crosslinkingsites in hologous helicases as described in Example 9, by sequencealignment and structural homology modeling, the correspondingcrosslinking target residues are identified in helicases with unknownstructures. Subsequently these helicases can be converted tosuperhelicase forms. Thus, in an embodiment, Rep-like thermophilichelicases featuring low or no cysteine content, and homologs ororthologs thereof are also suitable candidates for cross-linking to forma thermophilic superhelicase. Selected examples of thermophilicorthologs or homologs of UvrD, Rep and PcrA are shown in Tables 16-18.In certain exemplary embodiments, a suitable UvrD, Rep or PcrA helicaseis selected from the following species: Thermococcus sp. EXT9,Thermococcus sp. IRI48, Thermococcus sp. IRI33, Thermococcus sp. AMT7,Thermococcus nautili, Thermococcus onnurineus (strain NA1), Thermococcuskodakarensis (strain ATCC BAA-918/JCM 12380/KOD1) (Pyrococcuskodakaraensis (strain KOD1)), Thermococcus sibiricus (strain MM 739/DSM12597), Thermococcus paralvinellae, Thermus aquaticus Y51MC23, Thermusaquaticus Y51MC23, Thermus aquaticus Y51MC23, Thermus sp. RL, Thermussp. RL, Thermus sp. 2.9, Salinisphaera hydrothermalis C41B8, Thermusfiliformis, Meiothermus ruber, Thermus sp. NMX2.A1, Thermus thermophilusJL-18, Thermus scotoductus (strain ATCC 700910/SA-01), Thermusscotoductus (strain ATCC 700910/SA-01), Oceanithermus profundus (strainDSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strainDSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strainDSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strainDSM 14977/NBRC 100410/VKM B-2274/506), Oceanithermus profundus (strainDSM 14977/NBRC 100410/VKM B-2274/506), Thermus oshimai JL-2, Thermusoshimai JL-2, Thermus oshimai JL-2, Thermomonospora curvata (strain ATCC19995/DSM 43183/JCM 3096/NUMB 10081), Thermodesulfatator indicus (strainDSM 15286/JCM 11887/CIR29812), Geobacillus stearothermophilus (Bacillusstearothermophilus), Coprothermobacter proteolyticus (strain ATCC35245/DSM 5265/BT), Meiothermus silvanus (strain ATCC 700542/DSM9946/VI-R2) (Thermus silvanus), Anaerolinea thermophila (strain DSM14523/JCM 11388/NBRC 100420/UNI-1), Thermoanaerobacteriumthermosaccharolyticum M0795, Meiothermus ruber (strain ATCC 35948/DSM1279/VKM B-1258/21) (Thermus ruber), Meiothermus ruber (strain ATCC35948/DSM 1279/VKM B-1258/21) (Thermus ruber), Deinococcus radiodurans(strain ATCC 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB9279/R1/VKMB-1422), Thermodesulfobium narugense DSM 14796, Thermusthermophilus (strain HB8/ATCC 27634/DSM 579), Dictyoglomus thermophilum(strain ATCC 35947/DSM 3960/H-6-42), Thermus thermophilus (strainSG0.5JP17-16), Thermus thermophilus (strain SG0.5JP17-16), Thermusthermophilus (strain SG0.5JP17-16), Thermus sp. CCB_US3_UF1, Deinococcusgeothermalis (strain DSM 11300), Thermus thermophilus (strain HB27/ATCCBAA-163/DSM 7039), Thermus thermophilus (strain HB27/ATCC BAA-163/DSM7039), Marinithermus hydrothermalis (strain DSM 14884/JCM 11576/T1).

TABLE 16 Gene names Protein (primary)/Gene Entry (3D) Entry name namesOrganism Length encoded by L0B9N8 L0B9N8_9EURY UvrD Rep Thermococcus 591Plasmid helicase SFI sp. EXT9 pEXT9a L0B9J0 L0B9J0_9EURY UvrD RepThermococcus 547 Plasmid helicase SFI sp. IRI48 pIRI48 L0BAD9L0BAD9_9EURY UvrD Rep Thermococcus 591 Plasmid helicase SFI sp. IRI33pIRI33 L0BAT5 L0BAT5_9EURY UvrD Rep Thermococcus 591 Plasmid helicasesp. AMT7 pAMT7 W8NUG2 W8NUG2_9EURY Superfamily I Thermococcus 665 DNAand nautili RNA helicase and helicase subunits B6YXQ7 B6YXQ7_THEONUvrD/REP Thermococcus 533 helicase onnurineus (strain NA1) Q5JFK3Q5JFK3_THEKO DNA Thermococcus 661 helicase, kodakarensis UvrD/REP(strain ATCC BAA- family 918/JCM 12380/ KOD1) (Pyrococcus kodakaraensis(strain KOD1)) C6A075 C6A075_THESM DNA Thermococcus 716 helicase,sibiricus (strain UvrD/REP MM 739/DSM family 12597) W0I5I1 W0I5I1_9EURYDNA Thermococcus 659 helicase, paralvinellae UvrD/REP family proteinB7AA42 B7AA42_THEAQ DNA helicase Thermus 701 (EC 3.6.4.12) aquaticusY51MC23 B7A5I6 B7A5I6_THEAQ DNA helicase Thermus 868 (EC 3.6.4.12)aquaticus Y51MC23 B7A954 B7A954_THEAQ DNA helicase Thermus 542 (EC3.6.4.12) aquaticus Y51MC23 H7GEQ7 H7GEQ7_9DEIN DNA helicase Thermus sp.RL 1030 (EC 3.6.4.12) H7GH69 H7GH69_9DEIN DNA helicase Thermus sp. RL693 (EC 3.6.4.12) A0A0B0SAG4 A0A0B0SAG4_9DEIN DNA helicase Thermus sp.2.9 692 (EC 3.6.4.12) A0A084IL47 A0A084IL47_9GAMM ATP- Salinisphaera 670rep dependent hydrothermalis DNA helicase C41B8 Rep (EC 3.6.4.12)A0A0A2WMV1 A0A0A2WMV1_THEFI DNA helicase Thermus 665 (EC 3.6.4.12)filiformis A0A0D0N7B7 A0A0D0N7B7_MEIRU DNA helicase Meiothermus 706 (EC3.6.4.12) ruber W2U4X3 W2U4X3_9DEIN DNA helicase Thermus sp. 710 (EC3.6.4.12) NMX2.A1 H9ZQB5 H9ZQB5_THETH DNA helicase Thermus 693 (EC3.6.4.12) thermophilus JL-18 E8PM35 E8PM35_THESS DNA helicase Thermus708 pcrA1 (EC 3.6.4.12) scotoductus (strain ATCC 700910/SA-01) E8PL08E8PL08_THESS DNA helicase Thermus 621 pcrA2 (EC 3.6.4.12) scotoductus(strain ATCC 700910/SA-01 E4U8J8 E4U8J8_OCEP5 DNA helicase Oceanithermus719 (EC 3.6.4.12) profundus (strain DSM 14977/ NBRC 100410/ VKM B-2274/506) E4U4N5 E4U4N5_OCEP5 DNA helicase Oceanithermus 917 (EC 3.6.4.12)profundus (strain DSM 14977/ NBRC 100410/ VKM B-2274/ 506) E4UAI1E4UAI1_OCEP5 DNA helicase Oceanithermus 889 Plasmid (EC 3.6.4.12)profundus (strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506)E4UAI8 E4UAI8_OCEP5 DNA helicase Oceanithermus 638 Plasmid (EC 3.6.4.12)profundus (strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506)E4UAI4 E4UAI4_OCEP5 AAA ATPase Oceanithermus 606 Plasmid profundus(strain pOCEPR01 DSM 14977/ NBRC 100410/ VKM B-2274/ 506) K7QW32K7QW32_THEOS DNA helicase Thermus oshimai 693 (EC 3.6.4.12) JL-2 K7QWX5K7QWX5_THEOS DNA helicase Thermus oshimai 694 Plasmid (EC 3.6.4.12) JL-2pTHEOS01 K7QTS9 K7QTS9_THEOS DNA helicase Thermus oshimai 854 (EC3.6.4.12) JL-2 D1AF88 D1AF88_THECD DNA helicase Thermomonospora 799 (EC3.6.4.12) curvata (strain ATCC 19995/ DSM 43183/ JCM 3096/ NCIMB 10081)F8A884 F8A884_THEID DNA helicase Thermodesulfatator 503 (EC 3.6.4.12)indicus (strain DSM 15286/ JCM 11887/ CIR29812) A0A087LEB0A0A087LEB0_GEOSE Uncharacterized Geobacillus 807 proteinstearothermophilus (Bacillus stearothermophilus) B5Y6N2 B5Y6N2_COPPD DNAhelicase Coprothermobacter 696 pcrA (EC 3.6.4.12) proteolyticus (strainATCC 35245/DSM 5265/BT) D7BJL0 D7BJL0_MEISD DNA helicase Meiothermus 646Plasmid (EC 3.6.4.12) silvanus (strain pMESIL02 ATCC 700542/ DSM9946/VI- R2) (Thermus silvanus) E8MZN5 E8MZN5_ANATU DNA helicaseAnaerolinea 737 pcrA (EC 3.6.4.12) thermophila (strain DSM 14523/JCM11388/NBRC 100420/UNI-1) L0INW7 L0INW7_THETR ATP- Thermoanaerobacterium769 Plasmid dependent thermosaccharolyticum pTHETHE01 exoDNAse M0795(Exonuclease V), alpha subunit/ helicase superfamily I member D3PR99D3PR99_MEIRD DNA helicase Meiothermus 706 (EC 3.6.4.12) ruber (strainATCC 35948/ DSM 1279/VKM B-1258/21) (Thermus ruber) D3PLL2 D3PLL2_MEIRDDNA helicase Meiothermus 920 (EC 3.6.4.12) ruber (strain ATCC 35948/ DSM1279/VKM B-1258/21) (Thermus ruber) Q9RTI9 Q9RTI9_DEIRA DNA helicaseDeinococcus 745 (X-ray (EC 3.6.4.12) radiodurans crystallog- (strainATCC raphy (3)) 13939/DSM 20539/JCM 16871/LMG 4051/NBRC 15346/NCIMB9279/R1/VKM B-1422) M1E5C5 M1E5C5_9FIRM DNA helicase Thermodesulfobium610 (EC 3.6.4.12) narugense DSM 14796 Q5SIE7 Q5SIE7_THET8 DNA helicaseThermus 692 (EC 3.6.4.12) thermophilus (strain HB8/ ATCC 27634/ DSM 579)B5YD55 B5YD55_DICT6 DNA helicase Dictyoglomus 656 (EC 3.6.4.12)thermophilum (strain ATCC 35947/DSM 3960/H-6-12) F6DJA4 F6DJA4_THETG DNAhelicase Thermus 722 Plasmid (EC 3.6.4.12) thermophilus pTHTHE1601(strain SG0.5JP17-16) F6DIL2 F6DIL2_THETG DNA helicase Thermus 692 (EC3.6.4.12) thermophilus (strain SG0.5JP17-16) F6DJ67 F6DJ67_THETG DNAhelicase Thermus 1014 Plasmid (EC 3.6.4.12) thermophilus pTHTHE1601(strain SG0.5JP17-16) G8N9P8 G8N9P8_9DEIN DNA helicase Thermus sp. 704(EC 3.6.4.12) CCB_US3_UF1 Q1J014 Q1J014_DEIGD DNA helicase Deinococcus741 (EC 3.6.4.12) geothermalis (strain DSM 11300) Q745W4 Q745W4_THET2DNA helicase Thermus 551 Plasmid (EC 3.6.4.12) thermophilus pTT27(strain HB27/ ATCC BAA-163/ DSM 7039) Q72IS0 Q72IS0_THET2 DNA helicaseThermus 692 uvrD (EC 3.6.4.12) thermophilus (strain HB27/ ATCC BAA-163/DSM 7039) F2NK78 F2NK78_MARHT DNA helicase Marinithermus 716 (EC3.6.4.12) hydrothermalis (strain DSM 14884/JCM 11576/T1)

TABLE 17  SEQ ID  Entry Sequence NO:  L0B9N8MSEALPVTSFEFSLPEESVIKIYGPPGTGKTTTLVRIIEHLIGEHDHTEFLESYGLSLLFGQYGAEDV 58IFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAIDGVITQNFGSLSPEDWFRLFCRQNGLRFESSEMGYSNVFNDGNRLWNALTWAYNVYYPTKGPKARHEALKRLAPKLWKYPPLWEEYKTEKGILDYNDMLVKAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQFEIFRLLANYMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLKRSYRLPIVVQAKAMTVISKTRHRKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGVHFRHLKVDYRSIWEAGSKEWGTFRDLVQALLKARRGEELEIADLVTILYYSELIDWHLGEKLPEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKDDPFSVLDLEKIESLSPRHGKVAVELIREIMKEKSQVVSVPRDAEIYLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKHPFPVL L0B9J0MRVKIYGPPGTGKTTTLQRTIDYTLGNSSEPPIPLPESFPTDLEPKNLAFVSFTNTAIDVIGKRTGI 59TTRSKEAPYMRTIHGLILSVLAEHFDPVAVDNLGKLADIQAEFSMRMGYYYSKDPFEFAEGNMKFNVITRALELYLPKTGDVEEALKLIDNREDRKFALAWYRYKRQKKIMDFDDILVIGYEHLEDFYVPVEVAFIDEGQDNGPLDYILLEKGFEGAKFVFLAGDPLQSIYGFKGADPRLFVRWKADKEIILPRSYRLPKKVWLLSQSWALSLGIKGAVVRYAPSEKLGRVSRMKFIEALSYAVEQAKRGRSVLILARTNSLVKFVGNILSIEFGVAYGHLKRASYWESHLLKFIEGLQMLKLWDGVFPIKVQDTKPITGLIRKLKDKHAREVLRRWRDSRQWSLEVQAVLQRIKKNPSEYFYITDFDRQALKAYFSKARLDLTEELIIDTIHAAKGEEADVVIFLDFIPTRSEERINPEELQEKLVAYVGFTRAREELIIVPTPAIKYHPMRDFMGVRQILGVVNFHKHLLIKELVGGL L0BAD9MSEALPVTSFEFSLPRERIIKLYGAPGTGKTTTLVKIIEHLIGFQDHTEFLENYGINLPFGQYEPGE 60VIFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGLITQNFGSLSPEDWFRNFCRQNGLRFESSEMGYSNVFNEGNQLWNALTWAYNVYYPTKGPKARYEALKRLAPKLWKFPPLWEEYKKGRGILDYNDMLVRAYEGLRSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQFEIFRLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGLEWLRKSHRLPIVVQAKALTVISKTRHRKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGVHFEHLKVDYRSIWEAGSKEWGTFRDLVQALLKAKRGEELEVADLVTILYYSELIDWHLGEGISEKERYKKIAEQMEKTIEAIEKGLMPFDVLRVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQVVSIPKDARIYLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKHPFPVL L0BAT5MSEALSITSFDFTLPRERIIKIYGPPGTGKTTTLVRIIEHLIGFQDHTEFLENYGLSLPFGQYGAEDV 61IFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGLITQNFGSLSPEDWFRHFCRQNGLRFESSEMGYSNIFNEGNQLWNALTWAYNVYYPTKGPKARYEALKRLAPKLWKFPPLWEEYKKEKGILDYNDMLIRAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLQFEIFRLLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLSKSYRLPIVVQAKALTVISKTRHRKEKTVAPRTDLGDFKYKLFWFPDFLNDLVREAQEGHSIFILVRTNRQVLKLGKELILAGVHFEHLKVDYRSIWEAGSKEWGTERDLVQALLKAKKGEELEVADLVTILYYSELIDWHLGERISEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQVVSIPKDAKIYLDTLHASKGREADVVFLINDLPRKWSNILKTREELDAERRVWYVGLTRARKKVYLLNG KHPFPILW8NUG2 MNENEKLSKFIAKLKVLIEMERKAEIEAMRAEMRRLSGREREKVGRAVLGLNGKVIGEELGYFL62 VRYGREREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFITVALETVPEWALKSVRIDLYANDITFKRWLENLENLRESGRRALELYLGLREPEGGEEVEFTPFDKSLNASQRRAIAKALGSPDFFLIHGPFGTGKTRTLVELIRQEVARGNRVLATAESNVAVDNLVERLVDSGLKVVRVGHPSRVSRGLHETTLAYLMTQHELYGELRELRVIGENLKEKRDTFTKPAPKYRRGLTDRQILRLAEKGIGTRGVPARLIREMAQWLKINEQVQKTFDDARKLEERIAREIIREADVVLTTNSSAGLEVVDYGSYDVAIIDEATQATIPSVLIPINRAGREVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPGKSEMLTVQYRMNERLMEFPSREFYDGRIEADESIRRITLADLGVKSPEDGDAWAEVLKPENVLVFIDTARREDRFERQRYGSESRENPLEARLVKEAVEGLLRLGVKAEWIGVITPYDDQRDLISSLLPEEIEVKTVDGYQGREKEVIVLSFVRSNRKGELGFLKDLRRLNVSLTRAKRKLILIGDSSTLSSHPTYRRLVEFVRERETVVDAKRLIGKVKIK B6YXQ7MTAPIPTTYSILGVAGAGKTTQLIDLLNYLNFENSRNEKIWERHFEPVELNRIAFISFSNTAIQEIA 63NRTGIEIKARKKSAPGRYFRTVTGLAEVLLYENNLMTFEEVRSVSKLEGFRIKWAREHGMYYKPRDNDISYSGNEFFAEYSRLVNTYYHVKSLSEIIEMHSKSHLLLDYIREKEKLGIVDYEDILMRAYDYRNDIVVDLEYMIIDEAQDNSLLDYATLLPIAKNNATELVLAGDDAQLIYDFRGANYKLEHKLIERSEIILNLTETRRFGSEIANLATAIIDDMNYIQKREVLSAATHSTKVAHIDLFQMMSILQNMATTDLTVYILARTNAVLNYVAKVLDEYKIQYKKNERITDFDRFLLSLNRLMRNEYTNDDIYTIYNYLRNKVAREEELKERLFQHKLHWTEKDVLGILLLAYEQTTAKRILTTAKNTNFKIKLSTIHSAKGSEADVVELINSVPHKTKMKILENYEGEKRVLYVAVTRARKFLFIVDQPVARRYEQLYYIRSYESRAQGSLVNRVAVPVA Q5JFK3MNEKEVLLSKFIAHLKELVEMERRAEIEAMRLEMRRLSGREREKVGRAVLGLNGKVIGEELGYF 64LVRYGRDREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFLTVAIETVPEWALKGVRIDLYANDITFKRWMENLDNLRESGRKALELYLGLREPEESEPVEFQPFDKSLNASQRGAIAKALGSGDFFLVHGPFGTGKTRTLVELIRQEVARGHKVLATAESNVAVDNIVERLADSGLKVVRIGHPSRVSKALHETTLAYLITQHDLYAELRELRVIGENLKEKRDTFTKPAPKYRRGLSDREILRLAEKGIGTRGVPARLIREMAEWIRINQQVQKTFDDARKLEERIAREIIQEADVVLTTNASAGLEVVDYGEYDVAVID EATQATIPSVLIPINRAKREVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPEKSEMLIVQYRMNERLMEFPSREFYDGKIKAHESVKNITLADLGVSEPEFGNFWDEALKPENVLVFIDTSKREDRFERQRRGSDSRENPLEAKLVTETVEKLLEMGVKPDWIGVITPYDDQRDLISSMVGEDIEVKTVDGYQGREKEIIVLSFVRSNRRGELGFLTDLRRLNVSLTRAKRKLIAVGDSSTLSNHPTYRRFIEFVRERGTFIEIDGKKH C6A075MTRVQIPAGAPKYGPVAQPGQSARLISGRSGVRSPAGPPKALLKERFRELFIHKNPVITMHVK 65NYIAKLVDLVELEREAEIEAMREEMRRLKGYEREKVGRAILNLNGKIIGEEFGFKLVKYGRKEAFKTEIGVGDLVVISKGNPLASDLVGTVVEKGSRFIVVALETVPSWAFRNVRIDLYANDITFRRQLENLKKLSESGIRALKLILGKETPLKSSPEEFTPFDRNLNQSQKEAVSYALGSEDFFLIHGPFGTGKTRTLVELIVQEVKRGNKILATAESNVAVDNLVERLWGKVKLVRLGHPSRVSVHLKESTLAFQVESHERYRKVRELRNKAERLAVMRDQYKKPTPQMRRGLTNNQILKLAYRGRGSRGVPAKDIKQMAQWITLNEQIQKLYKFAEKIESEIIQEIIEDVDVVLSTNSSAALEFIKDAEFDVAIIDEASQATIPSVLIPIAKARREVLAGDHKQLPPTILSEEARALSETLFEKLIELYPFKAKMLEIQYRMNQLLMEFPSREFYNGKIKADGSVKDITLADLKVREPFFGEPWDSILKREEPLIFVDTSNRTDKWERQRKGSTSRENPLEALLVREIVERLLRMGIKKEWIGIITPYDDQVDSIRSIIQDDEIEIHTVDGYQGREKEIIILSLVRSNKKGELGFLMDLRRLNVSITRAKRKLVVIGDSETLVNHEYKRLIHFVKKYGRYIELGDTGIN W01511MNLIRYINHLKELVELEREAEIEAMREEMRKLTGYEREKVGRAVLGLNGKIIGEEFGYKLVKYGR 66KQEIKTEISVGDLVVISKGNPLASDLIGTVTEKGKRFLVVALETVPSWALRNVRIDLYANDITFKRQIENLDKLSESGKRALRFILGLEKPKESIDIEFKPFDEQLNESQKKAVGLALGSEDFFLIHGPFGTGKTRTVAEVILQEVKRGKKVLATAESNVAVDNLVERLWGKVKLVRLGHPSRVSKHLKESTLAYQVEIHEKYKRVREFRNKAERLAMLRDQYTKPTPQWRRGLTDRQILRLAEKGIGARGIPARVIKSMAQWITFNEKVQRLYNEAKKIEEEIVKEIIRQADVVLSTNSSAALEFIKDIKFIDVAVIDEASQATIPSVLIPIAKANKFILAGDHKQLPPTILSEEAKELSETLFEKLIELYPSKAKMLEIQYRMNERLMEFPSKEFYNGKIKAYDGVKNITLLDLGVRVFSFGEPWDSILNLKEPLVFVDTSKHPEKWERQRKGSLSRENLLEAELVKEIVQKLLRMGIKPESIGVITPYDDQRDLISLLLENDEIEVKTVDGYQGREKEVIILSFVRSNKKGELGFLTDLRRLNVSLTRAKRKLIAIGDSETLSAHPIYKRFVEFVKEKGIFVQLNQYVS QTSB7AA42 MGEAHPSEEALLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIARRGVFPSEIL67 AVTFTNKAAEEMKARLKAMVRGAGELWVSTFHAAALRILRVYGERVGLKPGFVVYDEDDQTALLKEVLKELGLAAKPGPIKSLLDRAKNQGVPPEHLLLELPEFYAGLSRGRLQDVLHRYQEALRAQGALDFGDILLYALKLLEEDGEVLKRVRKRARFIHVDEYQDTNPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIRNILDFTQDYPKARVYRLEDNYRSTEAILRFANAVIVKNALRLEKTLRPVKKGGEPVRLFRAESARDEAREVAEEIARLGPPFDRVAVLYRTNAQSRLLEQALASRGIPARVVGGVGFFERAEVKDLLAYARLSLNPLDAVSLKRVLNTPPRGIGPATVEKVQAIARERGLPLFEALKVAALTLPRPEPLRAFLALMEELMDLAFGPAEAFFRHLLLATDYPAYLKEAYPEDAEDRLENVEELLRAAKEAESLMDFLDKVALTARAEEPAEAEGRVALMTLHNAKGLEFPVVFLVGVEEGLPHRSSLSTQEGLEEERRLFYVGVTRAQERLYLSYAQEREIYGRLEPVRPSRFLEEVDEGLYEVYDPYRQSSRKPTPPPHRALPGAFRGGEKWEIPRFGPGTVVAAAGDEVTVHFEGVGLKRLSLKYADLRPA B7A5I6MRVYLASAGTGKTHALVEELKGLIQSGVPLRRIAALTFTRKAAEELRGRAKRAVLALSAEDPRLK 68EAEREVHGALFTTIHGEMAEALRFITAPLLSLDPDFALLDTFLAEALFLEEARSLLYRKGLDGGLARALLHLYRKRTLAETLHPLPGAEGVFALYLEALEGYRRRLPAELSPSDLEALALRILENPEALRRVVERFPHILLDEYQDTGPLQGRFFQGLKEAGARLVWGDPKQSIYLERNARVEVEREALKQAEEVRYLSTTYRHAQAVAEFLNRFTALFGEEGVRVRPHRQEVGRVEVHWVVGEGGLEEKRRAEAHLLLDRLMALREEGYAFSQMAVLVRSRSSLPPLEAAFRARGVPYALGRGRSFFARPEVRDLYHALRLSLLEGPPGPEERLALLAFLRGPWVGLDLSEVEEALKAQDPIPLLPEAVRAKLRALRALAGLPPLEALKRLSRDEAFLRRLSPRARVNLDALLLLAAMERFPDLEALLEWLRLRAEDPEAAELPEGEEGVQVLTVHGAKGLEWPWALFDLSRGENPKEEDLLVGLGGEVALRGTPAYKEVRKALRKAQAEEARRLLYVALSRARDVLIVTGSASGRPGPWVEALERLGLGPESQDPLVRRHPFKALPPLGDRPQTPPPPPLPAPYAHLAFPERPLPFVYSPSAFTKAKEPVPLAEALEKEALPEFYRALGTLVHYAIARHLDPEDEGAMAGLLLQEVAFPFAEGEKRRLLEEVRDLLRRYRGMLGPSLPPLEAREEDHAELPLVLPLGGTVWYGILDRLYRVGGRWYLEDYKTDREVRPEAYREQLAIYRRALLEAWGVEAEARLVYLRHGLVHPLDPEELERALKEGFPGMGPGEGGEKA B7A954MKGLTGSSRLRVYGPPGTGKTTWLKNEVERLLRSGVPGEEIAVCAFSRAAFREFASRLAGQVP 69EENLGTIHSLAYRAIGRPPLALTKDALSDWNRRVPDTWRVTPRVDGRGADLLDVMDPYEDEDSRPPGDKLYDRVAYLRNTLAPMAAWSEEERAFFQAWKSWMNAKGLVDEPGMLEAALAKPGGLGARFLLVDEAQDLTPLQLLLVEKWAQGARLALVGDDDQAIYGFMGADGASFLGVPVEDELVLGQSYRVPARVQRVAEAVIRRVQNRAPKRYAPRGDEGEVRLLWVPPEDPYHAWDALERVNRGESVLFLATAKYLLEELKRELLRVGEPYANPYAPHRHSFNLFPQGARSAWEKARSFLFPNRIAADVKAWTKHVSSKVFAVKGEEARRYIESFPDEEKVGDDHPIWNVFRPEHRPHAVGRDVSWLLDHLLGNAPKTMRQSLMVALKSPEAVLQGRARVWIGTIHSVKGGEADWVYVWPGYTRKAAREHPDQLHRLFYVAATRARKGLVLMDQGKAPHGYVWPRVDEFWGEVWV H7GEQ7MEANLYVAGAGTGKTYTLAERYLGFLEEGLSPLQVVAVTFTERAALELRHRVRQMVGERSLG 70HKERVLAELEAAPIGTLHALAARVCREFPEEAGVPADFQVMEDLEAALLLEAWLEEALLEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRXLRRRMEELFHGERPEERYPGFPKGWRXEEPEVVPDLLAWAGEVKFNKKPWLEYKXDPALXRLLKLLGGVKEGESPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEXEEVRAYYRGRERRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADPKVMERFQXEAAKEGLRVRRLEKSHRYHQGLADFHNRFFPPLLPGYGAVSAERKPEGEGPWVFHFQGDLEAQARFIAQEVGRLLSEGFQWDLGEKAYRPMSLRDVAVLGRTWRDLARVAEALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPXDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGTLDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVTLLTVHGAKGLEWPVVEVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEXGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAARDPEAARRRLLGEPEXEGGEAWXPXXPQETEEEVPGGAGVGRMTHALLERFEAXEDLEREGRAFLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAXAYLLQVGVYALALGKPRALVADLREGKLYEGASQQVEEKAEEVLRRLMGGEGQGRQPYPLAATDPGHGAPG H7GH69MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVTFT 71NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRXANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFXRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVELVGVEEGLLPHRNSLSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRXPKPXPPPHRPRPGAFRGGERVVHPREGPGTVVAAQGDEVTVHFEGXGLKRLSLKYAELXPA A0A0B0SAG4MDEALLSSLNEAQRQAVLHFQGPALVVAGAGSGKTRTVVHRVAYLIAHRGVYPTEILAVTFTN 72KAAEEMRERLKGMVRGAGEVWVSTFHAAALRILRVYGERVGLKPGFVVYDEDDQTALLKEVLKELGLSAKPGPIKALLDRAKNRGEPPEALLAELPEYYAGLSRRRLLDVFFRYQEALKAQGALDFGDILLYALRLLEEDQEVLARVRKRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILQFTADFPGAKVYRLEENYRSTEAILRFANAVIVKNALRLEKTLRPVKRGGEPVRLFRAKDAREEARFVAEEILRLGPPFDRIAVLYRTNAQSRLLEQALAGRGVGARVVGGVGFFERAEVKDLLAYARLALNPLDSVSLKRILNTPPRGIGPAIVEKVARLAQEKGLPLFEALKRAELLPRPEPVRHFVALMEELMDLAFGPAEAFFRHLLQATDYPAYLREAYPEDHEDRLENVEELLRAAKEAESLLDFLDKVALTARAEEPAGAEGKVELMTLHNAKGLEFPWELVGVEEGLLPHRNSLNTLEALEEERRLFYVGVTRAQERLYLSYAEEREVYGRLEATRPSRFLEEVEEGLYQEYDPYRSPRPVPPSHRPKPGAFKGGEKVVHPREGPGTVVAASGDEVTVHFEGVGLKRLSLKYADLRPA A0A084IL47MALPKLNPQQDAAMRYLDGPLLVLAGAGSGKTGVITRKIAHLIARGYDARRVVAVTFTNKAA 73REMKQRASKLISADDARGLTVSTFHSLGLQMIREEHAALGYKPRFSIFDSEDADKVLADLVGRDGDHRKATKAAISNWKSALIDPETATAQATGSDIPLARAYGEYQRRLKAYNAVDFDDLLALPVHLLSTDHEARERWQSRFRYLLVDEYQDTNAAQYEMMRLLAGARAAFTVVGDDDQSIYAWRGARPGNIADLSRDFPHLKVIKLEQNYRSVGNVLSAANQLIGASNQRAYEKTLWSAMGPGDRVRVIAAPDEAGEAERIASEISSHKLRLGTAYGDYAILYRGNFQSRAFEKALRERDIPYRVSGGRSFFERSEIRDLVTYLKLMVNPDDDAAFLRIVNLPRREIGPATLEALGRYAGSRHISLFDAARGIGLAGGVGERSGRRLADFVDWLRNLTQDSEGMTPRELVSQLIVDIDYRNWLRDTSANTKAARKRIENLDDFIGWLDRLDNAEDGKPVTLEDVVRRLSLMDFANQSEKDVENQVHLLTLHAAKGLEFDHVFLAGLEEGMLPHHACLEDDKIEEERRLLYVGITRARKTLALTYARKRRRGGEESDSVPSRFLEELPADELDWPSATGTRSKAANAEQGRDQVAALRAMLGASADS A0A0A2WMV1MPQVGFTDHFFKGLEALSREEQNRVREAVFAFMQDPKHPSFKLHRLEDIKTDRFWSARVSK 74DLRLILYHHPEMGWIFAYVGHHDDAYRWAETHQAEVHPKLGLLQIFRWEEVRVEPRKIKPLLPDYPDDYLLDLGVPPSYLKPLRLVETEDQLLGLIEGLPQDVQERLLDIAAGRPVTLPPKLAPSEEWFKHPLSRQHIHFIQNLDELRQALSYPWERWMVFLHPAQREAVERVFQGPARVTGPAGTGKTVVALHRAAALARRYPEEPLLLTTFNRFLASRLRSGLQRLLGEVPPNLTVENLHSLARRLHEQHVGPVKLVKEEDYGPWLLEAAQGLEYGKNFLLSEFAFADAWGLYTWEAYRGFPRTGRGVPLTARERLKLFGAFQKVWGRMENEGALTENGLLHRLRQRAEEGALPRFRAVVVDEAQDLGPAELLLVRALAQEAPDSLFFALDPAQRIYKSPLSWQALGLEVRGRSIRLKVNYRTTREIAKRAEAVLPKEV EGEMREVLSLLQGPEPEIRGFPTQEACQAELVRWLRWLLEQGVRPEEVAVLARVRKLAEGLAEGLRRAGIPVVLLSDQEDPGEGVRLGTVHSAKGLEFRAVALFGANRGLFPLESLLREAPSEADREALLAQERNLLYVAMSRARERWVGYWDEGSPFLTP A0A0D0N7B7MSDLLSSLNPSQREAVLHFEGPALVVAGAGSGKTRTVVHRIAYLLRERRVYPAEILAVTFTNKA  75AGEMKERLEKMVGRSARDLWVTTFHAAAVRILRTYGEYVGLKPGFVIYDEDDQNTLLKEVLK ELELEAKPGPFRSMIDRIKNRGAGLAEYMREAPDFIGGVPRDVAAEVYRRYQNSLRMQGALD FNDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYRLTRLLAGERPNLMVVGDPDQ SIYGFRNADINNILDFTKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTKPGGEPV RLYRAPNAREEAAFVAREIVKLGGYQQVAVLYRTNAQSRLLEEHLRRANVPVRLVGAVGFFER REIKDLLAYGRVAVNPDDSINLRRIVNTPPRGIGATTVARLVEHAQKTGITVFEAFRAAEQVISR PQQVQAFVRLLDELMEAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRAAQD WEEEEGGSLADDFLDSVALTAKAEEPQGGDAPVEATLMTLHNAKGLEFPTVFLVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERETYGKREYTRPSRFLQDIPQDLLKEVGAFGD GETRVLSQARPEPKPRTQPAEFKGGEKVKHPKFGSGTVVAAMGGEVTVMFPGVGLKRLAVK  FAGLERLEW2U4X3 MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVFPS 76 EILAVTFTNKAALEMRERLKRMVKGAGELWVSTFHSAALRILRVYGERVGLKPGFVVYDEDD QTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPV KPGGEPVRLYRARDARDEARFVAEEILRLGPPFDRVAVLYRTNAQSRLLEQALASRGVPARVVGGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALR VAAEVLPRPAPLRHFLALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPEDHEDRLENVEE LLRMKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHR SSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRA SAKVSPSPAPSEARASKPKPGAYRGGEKVIHPRFGQGTVVAAMGDEVTVHFEGVGLKRLSLK  YADLRPVGH9ZQB5 MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 77NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLEALLGELPEYYAGLSRGRLADVLVRYQEALKAQGALDFGDILLYALRLLKEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRLANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTSSRVEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDKVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVFLVGVEEGLLPHRNSLSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRVPKPAPPPHRPRPGAFRGGERVVHPRFGPGTVVAAQGDEVTVHFEGFGLKRLSLKYAELRPA E8PM35MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVFPS 78EILAVTFTNKAAEEMRERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKPGFVVYDEDDQTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEAREVAEEILRLGPPFDRVAVLYRTNAQSRLLEQTLASRGVPARVVGGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALRVAAEVLPRPAPLRHFLALMEELQELAFGPAEGFERHLLEATDYPAYLREAYPEDYEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPWFWGVEEGLLPHRSSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSPAPGEARASKPGAYRGGEKVIHPREGQGWVAAMGDEVTVHFEGVGLKRLSLKYA DLRPVGE8PL08 MLNPEQEAVANHFTGPALVIAGPGSGKTRTVVHRIARLIRKGVDPETVTAVTFTKKAAGEMR 79ERLVHLVGEETATKVETATFHSLAYHVLKDTGTVRVLPAEQARKLIGEILEDLQAPKKLTAKVAQGAFSRVKNSGGGRRELIALYTDFSPYIERAWDAYEAYKEEKRLLDFDDLLHQAVHELSTDIDLQARWQHRARFLIVDEYQDTNLVQFNLLRLLLTPEENLMAVGDPNQAIYAWRGADFRLILEFKKHFPNATVYKLHTNYRSHNGIVTAAKKVITHNTQREDLDLKALRNGDLPTLVQAQSREDEALAVAEVVKRHLDQGTPPEEIAILLRSLAYSRPIEATLRRYRIPYTIVGGLSEWNRKEVQLYLHLLQAASGNPESTVEVLASLVPGMGPKKARKALETGKYPKEAEEALQLLQDLRAYTGERGEHLASAVQNTLHRHRKTLWPYLLELADGIEEAAWDRWANLEEAVSTLFAFAHHTPEGDLDTYLADILLQEEDPEDSGDGVKIMTLHASKGLEFAVVLLPFLVEGAFPSWRSAQNPATLEEERRLFYVGLTRAKEHAYLSYHLVGERGATSPSRFARETPANLIHYNPTIGYQGKETDTLSKLAELF E4U8J8MSARDLLSSLNEQQQAAVQHFLGPALVIAGAGSGKTRTVVHRVAYLLAEREVYPAEVLAVTFT 80NKAAGEMRERLSRMVGRAAGELVVVSTFHSASLRILRRYGERIGLKPGFVVYDDDDQRVLLKEVLGSLGLEARPTYVRAVLDRIKNRMWSVDEFLAHADDWVGGLTKQQMAEVYARYQQRLAENNAVDFNDLLLRTIELFERHPEALEAVRQRARFIHVDEYQDTNPAQYRLTKLLAGDEANLMVVGDPDQSIYGERNADIQNILGFERDYRGAVVYRLEANYRSTAAILRVANALIERNQQRLEKTLRPVKPAGEPVRLYRAPDHREEAAFVAREVARLAGERALDDFAVLYRTNAQSRVLEEAFRRLNLPARIVGGVGFYERREVKDVLAYARLAVNPADDVALRRVINVPARGVGAASVGKLAAWAQAQGVSLLEAAHRAGELLAARQAAAVAKFTDLLTTLREAAEGTGPEAFLRLVLAETGYSEMLRREGDSEPRLENLEELLRAAAEWEEEHGGSVAEFLDEIALTARAEEPNAAPEKSVTLMTLHNAKGLEFPVVFWGVEEGLLPHRSSLGSDAEIEEERRLLYVGITRAQERLYLTLSEERETWGQRERVRPSRFLEEIPEDFLKPVGPFGDAHEPAPAPLSSAPVNRAAKGSASGRIGGEKVRHPRYGEGTVVATSGEGARQEVTVHFAEAGLKRLLVKYAGLERIE E4U4N5MKVRIASAGTGKTYALTSRFTAALAEHPPYRLAAVTFTRSAAAELKARLRERLLAIAAGREQPSG 81AEDVPPEAVVRRAGALATEVLGATVTTIHGFFAELLRQNALALGLEPDFLRIDASESQQIFAEEARAYVYLNEEDDALAEVLGRLFAKRSLAAELRPQGEAAEALWAHFRAVLERYARRLGGEALGPADIELHAWRLLERAGREEALAARIRSRLARVEVDEYQDTSPLQGRVFAALEALGVEVEVVGDPKQSIYAFRNADVEVEREAMRRGEPLPPLVTSWRHDRALVRFLNRYVDWVAEERPEAFARAEAPPVEARPDAGPGRVRLQLVQGEARQDALRPYEADQLARWLQERHAEHAWRDMAVLVRSHSSVPLLVRALAAHGLPHVVVGGRGFYDLIEVRDLVHAARVALDPRGRFSLAAFLRGPFAGLDLGRVERVLAAEDPLAELERAAPEVAERVDRLVRWVQTLRPLDFFERMVRTPFLEGASYLERLEPPARANVDQLLFKLASRRYGRLEFLLRDLEDLRGSDEAGVPEGGFDAVRIYTMHGSKGLEWPVVAVFDLNRGQPDGAEPFYVRPGSGEFAAEGDPDYPRFAAEWKERERQEAYRLLYVALSRPRSRLLLSLSVQLKPDGEGLRPKEWRRTLGRTLIEEMNLAAWDALEVERLDAARLPAPKAAAAAPRRAADVDERLRAPVEPLARPPWSPSALKAERPAPPELDDEGDVAVELEEPGVDPGLVARTVGILVHYAIGQDWGPERLQDLWNQEAVQRLTEPERTRVKTEVAQRLETYWRLLGTELPALDERDEDYAEFPLLLPTRTARLDIVWEGVIDRLYRVGDVWVLEDYKTDRELHPERYHFQLALYRRAVAAAWGIEPEARLVYLRFGEVVPLEAQLLEEAFERGTREAEEV E4UAI1MKVIVASAGTGKTTRLTQRYLEHLEQHPPQRVAAVTFTNKAAAELRERIFEALGRGSFYDFTPS 82PALAERLADYQVRVLEAPIGTIHSFFGYLLRLTAPMLGLDPHFEVIDPATARAWFLEEVRNLAIIEGAEVDETVTTALVELFKRRSISEAFEGTGDASRSLVAGFKKVYARWLTRLGGRYLDPSEIERRALALIRHPEALERVRSRLGVVLVDEYQDTAPIQARVFEALEEAGVPIEVVGDPKQSIYAFRDADVEGFREAHRRARENGNVETLTVSYRHPPALADFLNAFTSAEAALGKAFTAEEAPEVKPGREGDARVELITVTPGDGKATLDALRNGEARLLARELRRLHDEEGYDYGQMLVLFRRRHQLPPLLRALRGAGLPFAVVGLRGLYEEPEVRELYHALRLATGEAPRDSLAVFLSGPFGGLTLGQVREVLAQDAPESYLTLHHPEAAERLLRLRADAEKMRPAEALTRLIEAPTAKGPPFLDLLELEMADTVLYVLGRIEHTRTYPEAVATLESFRSGGEEEASLARLGGDAVRVMSAHAAKGLQAPVVVIFDADRTFNGNSDELVIEPRTGRVALNGEDAYESIAQALKARKEGEDHRLIYVALSRSSERLIVSAAVKEPRKGSWLHHLTEVLNLGSKFEHRNVTLAEIALEEPIEQEAATLPVDPELATPLPPAPPAVSSPTALKAERELEVPDPEEAWPADPEARLLGRIVGILVHEGIQRDWDPDDPEVLLALEGEQVLEEVPADRRPAVIEEVATLLRVYRTLLGSAIPSLEEREVDLAELPLVYPLGATAWEGVIDRLYRVGDVWYLEDYKTDREVHPERYHSQLALYREAVRKHWGIEPEVRLWLRTGQVVPLDAAALKEGLASYTGG E4UAI8MNEHERVIAHEVGPAAVVAGAGSGKTRAATLRAARLARTGERVGLVTFTASAAEEMRQRVL 83AEDVPAKHVWAGITHSLAFQILRQFPEAGGYEGFPEVLTPNDELRLFRRLWAELLDQDLDAELRRKLVKALGFFRKARAEEALEGWAARAGESLELDAEMLEALMISFQLRKREAGLASFDDLIEGASRALGDKDVRKWADRRFPFLIVDEYQDTSRAQETFLAALMPGEAPNLMVIGDPNQAIYGWRGAGSRTFERFQARYPQAVLYPLRKNYRSTRAVLRLAERAIARLYRSGQEAYYRLEGVKEEGEPPVLLTPPNAAAEATDVAREVARAVASGVPPEEIAVLARSSMQLAGVEDRLARLGVATRLLGGIRLSERREVICTLVQLIXAAWSLHERALVDFIEEAVPGLGERTLTRVEHAARPYNLVDRIMNDGAFVRGFSTRVQQGLFMTRTLLQLARATFEGVTGEAFAERFREFAQDLYGELLPGYLARIGKQGPNEEARRRHLEREVATVEAFAREEAEGGLDDLLARLAFLEQQDGPAVTLGTVHAVKGLEFEVVFWGMVEGAFPLADDSDPEEERRLFYVAATRAKRRLYLSAPTYGPRGKILQPSRYLEEALDEGLVRLQKVRPAA E4UAI4MVSEGRWKIERVVYLKDGFAVVAVRNEAGERHTAVGEMPTPVEGTWVRMETEHTVHPRY 84GPRLRVVRFLGLAPPPSKELAKIEGYLKLGFSEEAASWIAAREGSRPERAFDKPQELLVPGVPREVLRRVFPRLERLLGGLIDLLGEGHTAAPLFLLAERSGLGKEEIQELAREARKQRLIVEEQGRYGLVQPYRTERSIADGLLFRLKPGRGLRLTPPAGHGLSDEQARIFKLVRENRVVVLTGGPGSGKTTTIATLLAAPELHRMREGIAAPTGKAARRIAEVARLPAETIHRLLGLGEARRPLYHARNPLPYDLLVID ETSMLDAEIAAFLVDALAPSTSVIFVGDPDQLPPVGPGQFLRDLMTRVATLRLTQIFRQAQDSPIVNGAYALREGRMPLADGERLRLLPFEEEAAQTTLRTLLDELQRLEQIVGERPQVLVPGNRGPLGVRRLSPFLQQQLNPGGKPLGPIGWGMEAREGDPAVWIHNDYELGIMNGEVGVLRGGGSLGLTFETPTDRFAIPGNKRSRLVLAYAMTVHRSQGSEWPAVITILPKAHMALLSRELVYTALTRSKQYHTLLFHPEALYRARAVQASRRYTWLDVLLRG K7QW32MTAPGHPDALLAPLNPAQQEAVLHFQGPALVVAGAGSGKTRTVVHRVAYLMAHRGVYPGE 85ILAVTFTNKAAEEMKGRLKALVPGAGELWVATFHSAALRILRVYGEAIGLKPGRNYDEADQEALLKEVLKELGLSAKPGPLKALLDRAKNRGEAWEALEIPDYYAGLPKGKVLDVLRRYQEALRAQGALDFGDILVYALRLLEENPEVLAKVRKRARFIHVDEYQDTSPVQYRFARLLAGEEANLMAVGDPDQGIYSFRAADIRNILDFTRDFPGARVYRLEENYRSTEAILRFANAVIQKNRLRLEKTLRPVKPGGEPVRVYAAPEAREEAREVAEEIFRLGPPYERFAVLYRTNAQSRLLEQALAAKGLPYRVVGGVGFFERAEVKDLLAYARLSLNPEDGVSLKRVLNTPPRGIGPATLARLEALAQAEGVPLLGAIRLGAERFPKPEPLRAFLALLDELADLAFGPPEAFFRHLLSATDYLQYLKEHHPEDAEDRLENVEELLRAAKEAQDLQEFLDRVALTARADQDGGRGVALMTLHNAKGLEFPVVFLVGVEEGLLPHQSSLSTLEGLEEERRLFYVGVTRAQDRLYLSYAREREVYGRREPRRMSRFLEEVPEGLYLPHDPYRQGAQPKPAPRAQGAFRGGEKVVHPREGPGTVVAASGDEVIVHFEGVGLKRLSLKYADLRPA K7QWX5MASSLSKAELVPTPEQEKALHLYRSRQDFKLVAVAGSGKTTTLRLMAESFPRRHIAYLAFNRA 86MKEEARRKEPPNTRVFTLHALAYRRTVPGTPYEAKERLGNGQVRPVHVRERLQVDPLLAYVVRSGLERFIRSGDPEPLPRHLPRDWRKTVEARGPSGFAEVERAVKGVALLWKAMRDPKDPFPLSHDGYVRIWREEGAGGDPPAGVILVDEAQDLDPNFLTVLSGWRGKAQQVFVGDPRQQIYGWRGAVNAMGEIDLPESPLTVVSFRFGEPLASFVQAVTARQTQGLVPLVGRAGWATEVHVNLEPTPPFTILTRSNLGLVTALLEGAQLFSLQKEEAHWGGVEELVWLLTDLQAIKEGGERPRPHPELLGISKWEEVESLAEYSIVLNRLLRLAKEYDLEALAHKIAQLEIGPEEGAKLVLSTAHKAKGREWDRVLLWEDFYWVAAYRWFFPNTAPPPSEPSPEFLEEENIFYVAMTRARLGLHISLPEALAEEEAKRILDRLSQGVPSGEDRGEDERGETLPAPFTGPTPVSPKEATFPLPSLYDRLLSEALNGGRDPLLHLLRDDLARLSALSPTPLPPEVAQALWERARPEEALGAIREGLGAMWREDPYELLRAINALALLGGRNPRKLAKILGDRFPGGEEAEDLLFVARARKRELMGRSLAEFWRGLGASVRHPLLKAYARARS K7QTS9MRLYVASAGTGKTETLMGELKALLEGGVPLRRVAAVSFTRKSAEELRLRVRRLLEAHREAFWA 87REALREVHGALFTTLHGFMAEALRHTAPFLGLDPDFRVMDGFLAQALFLEEARSLLFLEGHPEAPELLELLEALYEKRSLAEAFTPLPGAEGLLALYERVLARYRARTQEVLGPGDLEAKALLLLRHPEALGRVAERFSHLLVDEFQDVNPLQGRFLRALEEAGVRWAVGDPKQSIYLFRNARVEVFLRARAAAEEVRALSRTHRHAKQVVELLNRFTTRFFRAEEGNRVEGVREAEGRVEVHWVLGKLEEARRAEARLLAQRLLALRAEGIPFGEMAVLVRARTSLPPLEKALRAAGVPFVRGRGQSFFARPEVRDLYHALRLALAERPYALEDRLSLLAFLRSPFLGLDLSELEEALRAEDPWPLLPKGVQEALEGLRALALLPPLEALRRLARDEGFLRRISRRARANLDTLLLLAAGAREPTLEDLLLWLALRAKDPESVELPEGGGGVTLLTVHGAKGLEWPWALYDVSRGPSERPPPLLVDEEGRVALKGTEAYRALLKEAERAEREEALRLLYVALSRARDLLLITGSTSQRPGPWAEALQALGLGPDAQDPWVETHPLEAIPPLPPIPQAPQDPRPAPYTPWRGEPRARPPWSPSAHLKAEAEPLEVLGEGEALPEWARAVGTLVHYAIARHLDPEDEGAMGGLLRQEVALAFGEGEREALLEEVRALLRAYRSLLSGALPPLEARAEDHAELPLLLPHKGTVWYGVLDRLYRVGDRWYLDDYKTDQKVRPEAYRFQLALYRKAVLEAWGVEAEARLVYLRHRQWPLSPAELEAALEGL D1AF88MSSSQVTGRPTIVKDAEIAVEQRRVDQAHARLEEMRAEAQAMIEEGYRQALAGTKGSLVDR 88DAM\NQAALRVQALNVADDGLVEGRLDLADGQTRYIGRIGVRTRDHEPMVIDWRAPAAEAFYRATPEDPQGWRRRVLHTRGR-R/VDLEDDLLDPSAADSLTIVGDGAFIASLARTREGTMRDIVATIQREQDEVIRAPADGTVLVRGAPGTGKTAVALHRVAYLLFRHRRRFGSRGVLWGPNRRFTAYIERVLPSLGEGSATLRSLGDLVEGVSATVHDPPELAALKGSAAMAPVLRRAVTDHPPGAPDKLRWHGGVWELGRPQLDKLRTSLHRRSTGSVNASRRRVAEALLDALWERYVHTGGTEPEPDEPVQGELALWEGILAEGGLAPLDEQDRPSSPADRTSREAFVKNVREQRAFTDFLTAWWPIRRPLDVLRSLGDAARLRRAAGRDLDRAQVELLAASWRRALAGDPPTLSYQDIALLDEIDALLGPPPQPSRATAREEDPYVVDGIDILTGEWADEDWEPGLQELTTTERLERARRVDDEVADVRPEYAHIWDEAQDLSPMQWRMLGRRGRQATWTIVEDPAQSAWEDLEEARKAMEAALDGPAARRGRSRRPRRRPRHEYELITNYRNTTEIAAVSARVLRLALPEARPARAVRSSGHRPVIDLVPEEELQAAARRAVRTLLECIVEGTIGVIVPLPGDAWGESDRRALSAGFPERVQVLDVLEAKGLEFDAAVICAPETIAAQSPRGLRVLYVAVSRATQRLTVLTADPWVRRRLAGGESAR F8A884MTSISLDQYQECtAVKAKGNTLWAGPGAGKTRVLLAKAIHLLEQGIDPEKVLILTFTIKTTQELK 89ERLASIGIKGVKVDTFHALAYDLLKAKGIKPRLATEEELKALARDLSKRKGLSLKDFRKALDKGENHYRSLWEEALKLHGLYDFSLLLKEATGHYLQQEKVYLLIDEFQDLNPELTSFLKTFTKAEFFLVGDPAQAIYGFRGACPQVIKEFVDYLAPQIYFLKKSYRVPEKVLNFAETLRETQGFPLEPLEAVQKGGNRLGLSFNKPFNEAKGVAKLVSELLGGLQMEASQRGLAPPEIAILSRVRTLLNPIKEAFIKFGIPFQVPSENLKEEISAIESLSDIAKSIKSLKELEAYLAEGPSSVKEAWLESQSLEGFLFRLEMLKTFASISIRKDGVPLLTIHEAKGLEFKWILVGAEDGLPFTLLEDYDLAEEKRVAYVAVTRAQESFYFTQVKTGRFLYGHKLSGKVSPFFETLPIKEKSSKTKPKARQKKLFG A0A087LEB0MTISVIDELLEKNKQNMNKTAKDAVEAQUAYAKKEVKKLQEIRPHPYFGRLDFEDEFGRETIYI 90GKKGLEKDGELIWDWRTDLGRLYNAYQGVQKTFQIGKENRPVTIHGKRGIVIKNGKVIKVTDIGKSEIIENDNGEKVKYMDDYLKEILTNTEEAHRLRDIIASIQAEQDEIIRLPLKDTIIVQGAAGSGKSTIALHRISYLLYQYHEQVKPKDILILAPNElFLSYIKDIVPEIElEGIEQRTFYDWASTYFTDVHDIPDLHEQYVHIYGSTEKEDLIKIAKYKGSLRFKKLLDDEVEYIGNTMIPHGDWIESGVILSKEEIHQFYHAKEHLPLNVRMKEVKEFIINWRNEQINIRKQQIEDEFEEAYRKWVVTLPEGEERKAVYEALEKAKQLRMKIFQEKMQHEISLIVKKMENIPALLMYKSVFQKKVFEKFHPDIDEELLSLLLKNGRQIKQERFMYEDIAPLIYLDAKINGKKLQYEHIVIDEAQDYSPFQLAIMKDYAKSMTILGDIAQGIFSFYGLDRWEEIESYVEKEKEFKRLHLQTSYRSTKQIMDLANRVLLNSNYDEPLVIPVNRPGDVPTIKKVESIGELYDEIVNSIRIFEEKGYKKIAILTASKQGAIDTYDQLMRRQITQMEVITEGHQALKEKIVIIPSYLVKGLEFDAVIIEDVSDETEKDETQHAKMLYMSITRAHHDLHLFYRGNISPLLEERDPSAPPKPRKSFADWLITDINDPYVEPQVEAVKRVKKEDMIRLFDDEEEEFVEEAFEDDRERYYDFHAWLKVWRRWAEMRKQLDEKS B5Y6N2MALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDLSKLNEAQKQAVTAPPKPLAIIAGP 91GSGKTRVLTYRALFAVKEWHLPPERILAITFTNKAADELKERLGRLIPEGDRIFAATMHSFAARMLRYFAPYAGISQNFVIYDDDDSKGLIEDILKQMNMDTKRFRPNDVLNHISAAKARMFDCNTFPEFIRQRYGSWGYYFDTVHQVFMTYERLKEQSQALDFDDLIMVLAQRMEDRPELREMIAGLFDLVMVDEFQDTNFAQYQMLLYMTNPHYSGMNNVTIVGDPDQSIYGFRAAEYYNIKRFIDDYNPEVVFLDLNYRSNRTIVDSASALINDSPSALFERKLESIKGAGNKLILRRPFDDADAAITAAFEVQRLFIKMGIPYEEIAVLMRTRALTARVEREFATRNIQYHIIGGVPFFARREIKDILAYLRLSRNAMDRVSLKRILTMKKRGFGTASLEKLFNFAEENKLTLLEAMKAAVESLLFKKLSMNDYLESLYTLIQTIQEIAEPSQAIYLVMEQENLLDHFRSISKSEEEYIERTENVKQLISIAEESADMDDFLQRSALGTRENNGGVEGVAISTVHGVKGLEFQAVILYYVTDGFFPHSLSVTTAEKEEERRLLYVAMTRAKEHLIFYVPYKQPWGNGFEQMARPSPFLRSIPKELWDGKPNEIESLYAPYSPQQKWSE D7BJL0MNDPIRHKEGPALVFAGAGAGKTRTLTQRVKWLVEEGEDPYSITLVTFTNKAAGEMKERIAR 92LVEAPLAEAVVNGTFHRFCLQSLQVYGREIGLEKVAVLDSAAQRKLAERIIAGLFPAKPPRGFTPMAALGAVSRAANSGWDDIQLATMYADLTEKIVNFRWAYEEAKKGLGALDYDDLLLRGVRLLKLSEGAARMVRRRAAYLMVDEFQDTNGVQLELVRAIAPGTSPNLMVIGDPDRSIYGWRGANYRTILEFRQHYPGAAVYGLYTNYRSQAGWEVANRIIAQNATRKPEMQEAHLPQSEEPFLLVAKNRWEEAHEVAQAVEFYRGQGIALEEMAVLMRANFLSRDLEQALRLRGIPYQFTGGRSFFERREIQLGMAVLKVLANPKDSLAVAALVEEMVEGAGPLGIQKVLEAAKAANLSPLEAFRNPAMVKGLRGKEVQAEAMRLAEVLQDQVARLAAEAPEYHALLKETLDRLGFEAWLDRLGEESEQ VYSRKANLDRLLQGMQEWQEVNPGAPLQDLVGTLLLEAGDTPAEEGQGVHLMTVHASKGMEFRVVFVIGLNEGLFPLSKASSSFEGLEEERRLMYVAVTRAKEVLHLSYAADGVVSRFAQEARVPVEEYDPRLGWSGRQNQQALKALLEIA E8MZN5MDSLEHLNPQQRAAVTASAGPVLVLAGPGSGKTRVLTFRIGYLLSQLGVAPHHILAVTFTNKA 93AREMQSRVEKLLGHSLQGMWLGTFHAICARILRREQQYLPLDANEVIEDEDDQQALIKRALRDLNLDEKLYRPTSVHAAISNAKNNLILPEDYPTATYRDEVVARVYKRYQELLVSSNAVDFDDLLLYAWKLLNEFSTVREQYARRFEHILVDEFQDTNLAQYELVKLLASYHRNLEVVGDEDQS1YRWRGADYRNVLRFEEDEPDRQKILLEQNYRSTQRVLDAAQAVINRNRNRTPKRLKSTPEHGEGEKLVLYEAVDDYGEAAFVVDTIQQLVAGGKARPGDFAIMYRTNAQSRLLEEAFLRAGVPYRLVGAMRFYGRREVKDMIAYLRLVQNPADEASLGRVINVPPRGIGDKSQLALQMEAQRTGRSAGLILMELGREGKDSPHWQALGRNASLLADEGSLLGEWHRLKDEISLPSLFQRILNDLAYREYIDDNTEEGQSRWENVQELLRIAYEYEEKGLTAFLENLALVSDQDTLPENVEAPTLLTLHAAKGLEFPIVFITGLDDGLIPHNRSLDDPEAMAEERRLFYVGLTRAKKRVYLVRAAQRSTYGSFQDSIPSRFLKDIPADLIQQDGRGRRMGRSWQSESRRSWDDNYAGDVIGSRPERAKPSHAPILQPREKPGMRVKHPSWGEGLWDSRIQDEDETVDIFFDSVGFKRVIASIANLEILS L0INW7MDINGQIIKLNRNKTQGTLKLTNGQKIKFKINSDSVKPIFLYEYYKFKGNMIEDTLIIDDIYGIAND 94ININDFTELEPSVAHDKINNICNRENVLHVGNLIDLINDENFITVVNDTIGEEKATIFLSNLQKIKDRQEYIDVWDIIKKTNPTEDINVPIKIVNALKYRASMNNITVSQLIKESPWIIEQLDIFDSITERKKIAENIATHYGLSNDSNKAVISYAIAMTNNYIQQGHSYIPYYTLVSRVSNSLKLDENKVNDTLKFLPNDNKSGYLIRDNKYKDEIENEYNSDKKIGYSVYLPKIEHMEKYIADIISSILKKKSTINKIELQKNLKLYRSENKLIFSKEQEEAIFSISDNKITVITGGAGTGKITVIKAIIDLVNKMGYTPWLAPTGIASQRVAPNVGSTIHKYARIFDTYDPVFDEIEENKENNSGKVIIVDEMSMITVPVFAKLLSVTLDADSFIFVGDPNQLPPIGAGGVFEALIELGNKNINNINTWLNQSFRSKNSIVKNAQN1LEDKPIYEDDNLNIIEAKSWNKIADEWNLIRKLLDNGVQYSDIMVLSSKRGEGKNGVSLLNERIRKEIENNKGKYAVGDIVITTRNDYDNKSSYFRSKELKKYINSIRHEERPTIENGTVGVIKDISDNEVIIEYNTPMPVEAKYNMEELDVVYIEYGFAITVHKAQGGQAKYIIFASDEPRNISREMLYTAITRCKNGKVFLIGGENEDWKIKKEHSFVLSKLKYRILDNIHQQEKESKINSKIVLINQ D3PR99MSDLLSSLNPSQQEAVLHFEGPALWAGAGSGKTRIVVHRIAYLLRERRVYPAEILAVITTNKA 95AGEMKERLEKMVGRPARDLWVSTFHAAAVRILRTYGEYVGLRPGFVIYDEDDQNTLLKEVLKELELEAKPGPFRAMIDRIKNRGAGLAEYMREAPDFIGGVPKDAAAEVYRKYQSGLRMQGALDENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYKLTRLLAGERPNLMWGDPDQSIYGERSADINNILDFTKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTRPGGEPVRLYRAPNAREEAAFVAREIVKLGNFQQIAVLYRTNAQSRLLEEHLRRANVPVRLVGAVGFFERREIKDLLAYGRVAVNPADSINLRRIVNTPPRGIGATTVSRLVEHAQKTGTTVFEAFRVAEQVISRPQQVQAFVRLLDEL1EAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRAAQDWEEEEGGSLSDFLDSVALTAKAEEPQGDAPAEAVTLMTLHNAKGLEFFIVELVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERFYGKREYTRPSRFLEDIPQDLLKEVGAFGDSEVRVLPQARPEPKPRTQLAEFKGGEKVRHPKEGSGTVVAAMGGEVIVMFPGVGLKRLAVK FAGLERLED3PLL2 MKVRVASAGTGKTASLVLRYLELIAKGTPLRRIAGVTFTRKAADELRVRVAAAIEEVLQTGRHLS96 EVASGGSRAAFQEAAREIAGATLSTIHGEMAQCLRLAAPLHLDPDFSMLGDWEAQAIFEEEWQTLRYLAQDAHHPLFGLVSDELTEPLLHLFSRRSQAEVFEPAAGEANQHLLQVYQTVYAAYEARLGANLLSPSELERKALELARNDRAMKRVLERVRVLLVDEYQDVNPVQGAFFAALEQARLPIEIVGDPKQSIYAFRNADVSVERKALREGKSEPPLTHSYRHSRVLVRELNGLIGYLAKEGLGEGLEEAPPVEGVRPEQGRLEVHWVVGELPLEELRKQEARVLAGRLAALRGPIEYSQMAVLVRSYGSVRFLEEALAEAQIPYVLLQGRGYYERQEVRDLYHALRAALDPRGLSLAVFLRSPFGQHTEAGPLKPLELPQIEGVLRADDPLGRLAQHWPSVYERLRQIQAQVRLMAPLEVLKFLIRAPLMDGRPYHDFLEPRARENVDALLFYFAPRPPQNLEGLERLELLSRQADAGDVPQSGEGVQILTVHQAKGLEWPLVAVFDLGRMNVHRPQPLYLGQGPNGGDGGRLRRVVVALPETPQFEAFRQQVKLQEEEESYRLYVAASRARDTLLTASASHGQPEGWGKVLEAMNLGPASKPYHRPDFHLQTWPYQPAPPVRVLSQPAPLQPSPWVDARFEPEPFPPLESPSALKRLEAEPLPLPDPEEGEAVPGRARAIGTLVHYAIGQNWRPDNPQHLANLEAQEVMFPFGPDERRGIMAEVQALLEHYQELLGRALPWPRDEDYPEFAVALPLGSTVWQGVIDRLYRVGQQWYLEDYKTDQEMRPERYLVQLGIYLAAIRQAWQIEPEVRLVYLRFGWVERLDKAILEAALGEIMPKGEGLRR Q9RTI9MTSSAGPDLLQALNPTQAQAADHFIGPALVIAGAGSGKTRTLIYRIAHLIGHYGVHPGElLAVT 97FINKAAAEMRERAGHLVPGAGDLWMSTFHSAGVRILRTYGEHIGLRRGFVIYDDDDQLDI1KEVMGSIPGIGAETQPRVIRGIIDRAKSNLWTPDDLDRSREPFISGLPRDAAAEAYRRYEVRKKGQNAIDEGDLITETVRLFKEVPGVLDKVQNKAKFIHVDEYQDTNRAQYELTRLASRDRNLLWGDPDQSIYKFRGADIQNILDFQKDYPDAKVYMLEHNYRSSARVLEAANKLIENNTERLDKTLKPVKEAGQPVTFHRATDHRAEGDYVADWLTRLHGEGRAWSEMAILYRTNAQSRVIEESLRRVQIPARIVGGVGFYDRREIRDILAYARLALNPADDVALRRIIGRPRRGIGDTALQKLMEWARTHHTSVLTACANAAEQNILDRGAHKATEFAGLMEAMSEAADNYEPAAFLRFVMETSGYLDLLRQEGQEGQVRLENLEELVSAAEEWSQDEANVGGSIADFLDDAALLSSVDDMRTKAENKGAPEDAVTLMTLHNAKGLEFPWFIVGVEQGLLPSKGAIAEGPSGIEEERRLFYVGITRAMERLLMTAAQNRMQFGKTNAAEDSAFLEDIEGLFDTVDPYGQPIEYRAKTWKQYRPTVPAATTAVKNTSPLTAELAYRGGEQVKHPKFGEGQVLAVAGVGERQEVTVHFASAGTKKLMVKFANLTKL M1E5C5MDLNLNEDQKRAVYSDSRALLIVAGAGTGKTRVLITRAARLIKENPDARYLLLTFTKKAAREM 98TTRVRELIEEDTKNRLYSGTEHSFCSNIIRRRSERVGLTNDEVIIDESDSLDLMKKVESRIYSKEKID SLIFKPKDILSLYSYARNNNQDFIEIVQRKYKYVNFEDIKKIISLYELNKKERNYLDFDDLLMYGLLAIKTLEKSPFDEVLVDEFQDTNQIQAEMLYYFYDLGSRISAVGDDAQSIYSFRGAYYENMENFIKRLDAEKIILSSNYRSTQQILDIANSIIQSSYSSIKKELVANVRLKENVKPKLVIVSDDWEEARYVAREMQKFGEKGLKVAALYRAAYIGRNLESQLNSMGIKYSFYGGQKLTESAHAKDFIVISFLRVEVNPKDEIALIRILKMFPGIGEKKAEKIKDAVISGDNLKKALSKEKNLEELNIFFDKLFKITDWHDLLELVFDFYKDIMNRLYPENYEEREEDLIKFMDMSSNYDNLVEYLEAFTLDPVEKSEEDNNNVILSTIHSAKGLEFDWELLSVIESVYPHFRAQSTDEIEEERRLFWAITRAKQRLIFTFPRHSKKSRGYFAKNTISPFLREKDNYLEVFIAR Q5SIE7MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 99NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLIAGEEANLMAVGDPDQGlYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFSRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGLEFPWELVGVEEGLLPHRNSVSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERWHPREGPERIVAAQGDEVIVHFEGFGLKRLSLKYAELKPA B5YD55MNNQFDSEKKIFIIPSRKKKEFLERIEKDLNEEQRKWLEADGPSLVIAGPGSGKTRTIVYRVGYL 100VALGYSPKNIMLLTFINQAARHMINRMALIRESIEEIWGGTEHHVGNRILRVYGKIIGINEQYNILDREDSLDLIDECLEELFPEENLGKGILGELFSYKVNTGKNWDEVLKIKAPQIIDKIEIVQKVFERYEKRKRELNVLDYDDLLFFWYRLLLESEKTRKILNDRFLYILVDEYQDTNWLQGEIIRLTREENKNILWGDDAQSIYSFRGATIENILSEPEIFPGTRIFYLVENYRSTPEIINLANEIIKRNTRQYFKEIKPVLKSGSKPKLVVVVRDDEEEAQFWEVIKELHKEGVKYKDIGVLERSNYHSMAVQMELTLQGIPYEVRGGLRFFECtAHIKDMISLLKILFNPQDEISAQRFFKLFPGIGRAYAKKLSQVLKESKDFDKIFQMQFSGRTLEGLRILKNIWDKIKVIPVQNFSEILRVFFNEYYKDYLERNYPDFKDREKDVDQLILLSERYDDLEKELSELTLYTYAGEKLLEEEEEEKDEWLSTIHQAKGLEWHAVFILRLVQGDFPSYKSMDNIEEERRLFYVAVTRAKRELYVITYLTRKVKDMNVFTKPSIFLEELPYKELFEEWIVQBEIMLSPEGGEEETKAIPLEEEILLAWRVESAALPPNFLAPVSASLHTLVREAEGKEGAELEAYAWER F6DJA4LEELARTSWKDAIQSFLEVAAEKPEVLRAGLLWERTWNRLSPEEREALYRKAERFKPTAELASK 101ASFLQGPPPPPKPLSPSVQAARSSPPRFTPTPEQEEAVRAFLSREDMKLVAVAGSGKIIILRLMAQSAPKERLLYVAFNRSVRDEAERTFPGNVEVLTLHGLAHRHWRGSGAYQRKLAARNGRVTPGDVLEALELPRERYALAYVIRSTLEAFLRSASEVPTPAHIPPEYREVLQRRDKDPFSERYVLKAVRLIWKLMQDPDDSFPLSEDGFVKIWAQAGAKIRGYDAVLVDEAQDLSPVFLQVLEAHRGELRRVYVGDPRQQIYGWRGAVNAMDKLDAPERKLTWSFREGEDIARGVRRFLAHVGSPIELHGKAPWDTEVSLARPEPPYTALCRTNAGAVEAVISELLEEGREGARVFWGGVDEIAWLLRDAHLLKVGGEREKPHPELALVENWEELEELAKEVNHPQARMLVRLARRYDLLELARLLKHAQADEEGKADLWSTLHKAKGREWDRWLWGDFIPVWDEKVREFYRKQGALDELKEEENVVYVALTRARRFLGLDQLPDLHERFFQGEGLVKPPSVSPLSVGGAGVSADLLRELEVRVLAKLEDRLKEVAEVLAALLVEEASKAVAEAMREMGLLGEEG F6DIL2MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTWHRVAYLVARRGVFPSEILAVTFT 102NKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFWYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFPRAEPLRHFVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPWFLVGVEEGLLPHRNSLSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERWHPRFGPGTVVAAQGDEVTVHFEGVGLKRLSLKYAELKPA F6DJ67MEANLYVAGAGTGKTYTLAERYLGFLEEGLSPLQWAVTFTERAALELRHRVRQMVGERSLG 103HKERVLAELEAAPIGTLHARVCREFPEEAGVPADFQVMEDLEAALLLEAWLEEALLEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRALRRRMEELFHGERPEERYPGFPKGWRTEEPEWPDLLAWAGEVKFNKKPWLEYKGDPALERLLKLLGGVKEGFSPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEREEVRAYYRGRERRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQS1YSFRRADPKVMERFQAEAAKEGLRVRRLEKSHRYHQGLADFFINRFFPPLLPGYGAVSAERKPEGEGPWVFEIFQGDLEAQARFIAQEVGRLLSEGFQVYDLGEKAYRPMSLRDVAVLGRIWRDLARVAEALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPKDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGTLDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVFLLTVHGAKGLEWPWFVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEEGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPIAARDPEAARRRLLGEPEPEGGEAWEPDGPQETEEEVPGGAGVGRMTHALLERFEAPEDLEREGRAFLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAKAYLLQVGVYAIALGKPRALVADLREGKLYEGASQQVEEKAEEVLRR LMGGDRPEAG8N9P8 MDAFPSGKPLDEAWLSSLNEAQRCtAVLHFEGPALWAGAGSGKTRIVVHRVAYLMARRGV 104YPSEILAVITTNKAAEEMRERLKAMVKGAGELWVSTFHAAALRILRFYGERVGLKPGFVVYDEDDQTALLKEVLKELGVSAKPGPIKALLDRAKNRGEPPERLLADLPEYYAGLSRGRLLDVLHRYQQALWAQGALDFGDILLLALKLLEEDPEVRKRVRKRARFIHVDEYQDTSPVQYRLTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILQFTEDFPGAKVYRLEENYRSTERILRFANAVIVKNALRLEKTLRPVKSGGEPVRLFRARDAREEARFVAEEVLRLGPPYDRVAVLYRTNAQSRLLEQALASRGIGARWGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATVEKVQA1AQEKGLPLYEALKVAAQVLPRPEPLRHFLALMEELMDLAFGPAEAFFRHLLEATDYPAYLKEAYPEDLEDRLENVEELLRAAREAEGLMDFLDKVALTARAEEPGEAGGKVALMTLHNAKGLEFPWFLVGVEEGLLPHRSSVSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRPEASRPSRFLEEVEEGLYEEYDPYRLPPPKPVPPPHRAKPGAFRGGEKWHPREGLERNAASGDEVIVHFDGVGLKRLSLKY ADLRPAQ1J014 MPDLPASSLIAQLNPNQAQAANHYTGPALVIAGAGSGKTRTLVYRIAHLIGHYGVDPGEILAV105 TFTNKAAAEMRERARHLVEGADRLWMSTFHSAGVRILRAYGEHIGLKRGFVIYDDDDQLDILKEIMGSIPGIGAETHPRVLRGILDRAKSNLLTPADLARHPEPFISGLPREVAAEAYRRYEARKKGQNAIDEGDLITETVRLFQEVPAVLERVQDRARFIHVDEYQDTNKAQYELTRLLASRDRNLLWGDPDQSIYRFRGADIQNILDFQKDYLDAKVYMLEQNYRSSARVLTIANKLIENNAERLEKTLRPVKEDGHPVLFHRATDQRAEGDFVAEWLTRLHAEGMRFSDMAVLYRTNAQSRVIEESLRRVQIPAKIVGGVGFYDRREIKDVLAYARLAINPDDDVALRRIIGRPKRGIGDTALERLMEWARVNGTSILTACAHAQELNILERGAQKAVEFAGLMHAMSEAADNDEPGPFLRYVIETSGYLDLLRQEGQEGQVRLENLEELVSPAEEWSRENEGTIGDFLDDAALLSSVDDMRTKQENKDVPEDAVTLMTLHNAKGLEFPWFIVGTEEGLLPSKNALLEPGGIEEERRLFYVGITRAMERLFLTAAQNRMQYGKTLATEDSRFLEEIKGGFDIVDAYGQVIDDRPKSWKEYRPTESARPGAVKNTSPLTEGMAYRGGEKVRHPKFGEGQVLAVAGLGDRQEVTVHFPSAGTKKLLVKFANLTRA Q745W4MALRPTEEQLKAVEAYRSGQDLKWAVAGSGKITTLRLMAEATPGKRGLYLAFNRSVQQEA 106ARKFPRNVRPYTLHALAFRMAVARDEGYRAKFQAGKGHLPAQAVAEALGLRNPLLLHAVLGTLEAFLRSEAASPDPGMIPLAYRTLRAGTKTWPEEEAFVLRGVEALWRRMTDPKDPFPLPHGAYVKLWALSEPDLSFAEALLVDEAQDLDPIFLKVLEAHRGRVQRVYVGDPRQQIYGWRGAINAMDRLEAPEARLTWSFRFAETLARFVRNLTALQDRPVEVRGKAPWATRVDAALPRPPFTVLCRTNAGWGAWVTHEVHRGRVHWGGVEELVHLLRDAALLKKGEKRTDPHPDIAMVETWEELEALAEAGYAPAYGVLRLAQEHPDLEALAAYLERAWTPVEVAAGWVSTAHKAKGREWDRVVLWDDFYPWWEEGWRVNWGSDPAHLEEENLLYVAATRARKHLSLAQIRDLLEAVDRMGVYRVAEEATRAYLLLSAEVLRGVATDPRVPAEHRVRALKALGYLERGEEALDSPGKPGGQG Q721S0MSDALLAPLNEAQRQAVLHFEGPALWAGAGSGKTRTVVHRVAYLVARRGVEPSEILAVTFT 107NKAAEEMRERLRGLVPGAGEVVVVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEAREVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARWGGVGFFERAEVKDLLAYARIALNPLDAVSLKRVLNTPPRGIGPATWARVQLIAQEKGLPPWEALKEAARTFPRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGLEFPWFLVGVEEGLLPHRNSVSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVHPREGPGTVVAAQGDEVEVHFEGFGLKRLSLKYAELKPA F2NK78MDLLRDLNPAQREAVQHYTGPALVVAGAGSGKTRIVVHRIAYLIRHRGVYPTEILAVTFTNKA 108AGEMKERLARMVGPAARELWVSTFHSAALRILRVYGEYIGLKPGFVVYDEDDQLALLKEVLGGLGLETRPQYARGVIDRIKNRMWSVDAFLREAEDWVGGLPKEQMAAVYQAYEARMRALGAVDFNDLLLKVIGLFEAHPEVLHRVQQRARFIHVDEYQDTNPAQYRLTRLLAGAERNLMVVGDPDQSIYGFRNADIHNILNFEKDYPDARVYRLEENYRSTEAILRVANAVIEKNALRLEKTLRPVRSGGDPVFLYRAPDHREEAAFVAREVQRLKGRGRRLDEIAVLYRTNAQSRVLEEAFRRQNLGVRIVGGVGFYERREVKDVLAYARAAVNPADDLAVKRVLNVPARGIGQTSLAKLSQLAETARVSFFEALRRAGEVLARPQAQAVQREVALIEGLANAAYDTGPDAFLRLVLAETGYADMLRREPDGEARLENLEELLRAAREWEEQHAGTIADFLDEVALTARAEEPEGEVPAEAVTLMTLHNAKGLEFPVVFIVGVEEGLLPHRSSTARVEDLEEERRLFYVGITRAQERLYLTLSEERETYGRREAVRASRFLEDIPEAFLQPLSPFGEPLGAGREPVAVRPTRRSSAAGGFRGGEKVRHPRFGQGLVVAASGEGDRQEVTVHFAGVGLKKLLVKYAGLERIE

TABLE 18  >tr|L0B9N8|L0B9N8_9EURY UvrD Rep helicase SFIOS = Thermococcussp. EXT9 GN = e9a-1 PE = 4 SV = 1 (SEQ ID NO: 58)MSEALPVISFEFSLPEESVIKIYGPPGTGKTTILVRIIEHLIGETHDHTEFLESYGLSLLFGQYGAEDVIFMTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAIDGVITQNFGSLSPEDINFRLFQRQNGLRFESSEMGYSNVENDGNRLWNALTWAYNVYYPTKGPKARHEALKRLAFKLWKYPPLWEEYKTEKGILDYNDMINKAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLUEIERLLANYMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLKRSYRLPIVVQAKAMTVISKTRHRKEKTVAPRIDLGDFKYKLFWEPDFLNDLVREAUGHSIFILVRTNRQVLKLGKELILAGVHFRHLKVDYRSIWEAGSKEWGTERDLVQALLKARRGEELEIADLVTILYYSELIDWHLGEKLPEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKDDPFSVLDLEKIESLSPRHGKVAVELIREIMKEKSQVEVPRDAEITLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKEPFPVL >tr|L0B90|L0B9J0_9EURY UvrD Rep helicase SFIOS =Thermococcus sp. IR148 GN = 148-1 PE = 4 SV = 1 (SEQ ID NO: 59)MRVKIYGPPGIGKITTLQRTIDYTLGNSSEPPIPLPESEPTDLEPKNLAFVSFINTAIDVIGKRTGITTRSKEAPYMRTIHGLILSVLAEHFDPVAVDNLGKLADIQAEFSMRMGYYYSKDPFEFAEGNMKENVITRALELYLPKTGDVEEALKLIDNREDRKFALAWYRYKRQKKIMDFDDILVIGYEHLEDFYVPVEVAFIDEGQDNGPLDYILLEKGFEGAKFVFLAGDPLQSIYGFKGADPRLEVRWKADKETILPRBYRLPKKVWLLSQSWAISLGIKGAVVRYAPSEKLGRVSRMKFIEALSYAVEQAKRGRSVLILARTNSLVKFVGNILSIEFGVAYGHLKRASYWESHLLKFIEGLQMLKLWDGVTPIKVQDTKPITGLIRKLKDKHAREVLRRWRDSROISLEVQAVLQRIKKNPSEYFYITDFDRQALKAYFSKARLDLTEELIIDTIHAAKGEEADVVIFLDFIPTRSEERINPEELQEKLVAYVGFTRAREELIIVPIPAIKYEPMRDFMGVRQILGVVNEHKHLLIKELVGGL >tr|L0BAD9|L0BAD9_9EURY UvrD Rep helicase SFIOS = Thermococcussp. IRI33 GN = i33-1 PE = 4 SV = 1 (SEQ ID NO: 60)MSEALPVTSFEFSLPRERIIKLYGAPGTGKTTILVKIIEHLIGFUHTEFLENYGINLPFGWEPGEVIENTFQTSALKEFEARTGIKVKDRQNKPGRYYSTVHGIAFRLLIDSGAVDGLITQNFGSLSPEDINFRNFCRQNGLRFESSEMGYSNVENEGNQLWNALTWAYNVYYTTKGPKARYEALKRLAFKLWKEPPLWEEYKKGRGILDYNDMTVRAYEGLRSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLUEIFRLLANHMDLVTIAGDDDQTIFSYQGADPRLMNYVPGLEVVLRKSHRLPIVVQAKALTVISKTRHRKEKTVAPRIDLGDFKYKLFWFPDFLNDLVREAUGHSIFILVRTNRQVLKLGKELILAGVHFEHLKVDYRSIWEAGSKEWGTERDLVQALLKAKRGEELEVADLVTILYYSELIDWHLGEGISEKERYKKIAEQMEKTIEAIEKGLMPFDVLRVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQVVEIPKDARIYLDTLHASKGREADVVFLINDLPRKWSSILKTREELDAERRVWYVGLTRARKKVYLLNGKEPFPVL >tr|L0BAT5|L0BAT5_9EURY UvrD Rep helicase OS =Thermococcus sp. AMT7 GN = a7-1 PE = 4 SV = 1 (SEQ ID NO: 61)MSEALSITSFDFTLPRERIIKIYGPPGTGKTTILVRIIEHLIGFUHTEFLENYGLSLPFGQYGAEDVIFMTRNSALKEFEARTGIKVKDRQNKPGRYYSTVEGIAFRLLIDSGAVDGLITQNFQSLSPEDWFRHFCRQNGLRFESSEMGYSNIFNEGNQLWNALTWAYNVYYTTKGPKARYEALKRLAFKLWKETPLWEEYKKEKGILDYNDMLTRAYEGLKSGEIDPRNLPGHKYSPKVLIVDEFQDLSPLUEIFRLLANHMDLVIIAGDDDQTIFSYQGADPRLMNYVPGREIVLSKSYRLPIVVQAKALTVISKTRHRKEKTVAPRIDLGDFKYKLFWFPDFLNDLVREAUGHSIFILVRTNRQVLKLGKELILAGVHFEHLKVIDYRSIWEAGSKEWGTFRDLVQALLKAKKGEELEVADLVTILYYSELIDWHLGERISEKERYKKIAEQMEKTIEAIEKGLMPFDILKVKENPFSVLDLEKIESLSPRHGKVAVELIKELMKEKSQNEIPKDAKITLDILHASKGREADVVFLINDLPRKWSNILKTREELDAERRVWYVGLTRARKKVYLLNGKHPFPIL >tr|W8NUG2|W8NUG2_9EURY Superfamily IDNA and RNA helicase andhelicase subunits OS = Thermococcus nautili GN = BDO1 1302 PE = 4 SV =1 (SEQ ID NO: 62)MNENEKLSKFIAKLKVL1EMERKAEIEAMRAEMRRLSGREREKVGRAVLGLNGKV-IGEELGYFLVRYGREREIKTEISVGDLVVISKRDPLKSDLVGTVVEKGKRFITVALETVPEWALKSVRIDLYANDITFKRWLENLENLRESGRRALELYLGLREPEGGEEVEFTPFDKSLNASQRRAIAKALGSPDFFLIHGPFGTGKTR7LVELIRQEVARGNRVLATAESNVAVDNLVERIVDSGLKVVRVGHPSRVSRGLHETTLAYLMTQHELYGELRELRVIGENLKEKRDIFTKPAPKYRRGLTDRQILRLAEKGIGTRGVPARLIREMAQWLKINEQVQKTFDDARKLEERIAREIIREADVVLTTNSSAGLEVVDYGSYDVAIIDEATQATIPSVLIPINRAGRFVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPGKSEMLIVQYRMNERLMEFPSREFYDGRIEADESIRRITLADLGVKSPEDGDAWAEVLKPENVIVFIDTARREDRFERQRYGSESRENPLEARLVKEAVEGILRLGVKAEWIGVITPYDDQRDLISSLLPEEIEVKTVDGYQGREKEVIVLSEVRSNRKGELGELKDLRRLNVSLTRAKRKLILIGDSSILSSHPTYRRIVEFVRERETVVDAKRIIGKVKIK >tr|B6YXQ7|B6YXQ7_THEONUvrD/REPhelicase OS = Thermococcusonnurineus (strain NA1) GN = TON_1380 PE = 4 SV = 1 (SEQ ID NO: 63)MIAPIPTTYSILGVAGAGKITQLIDLLNYLNFENSRNEKIWERHFEPVELNRIAFISFSNTAIQEIANRIGIEIKARKKSAPGRYFR7VTGLAEVLLYENNLMTFEEVRSVSKLEGFRIKWAREHGMYYKPRDNDISYSGNEFFAEYSRLVNTYYHVKSLSEIIEMHSKSHLLLDYIREKEKLGIVDYEDILMRAYDYRNDIVVDLEYMIIDEAUNSLLDYAILLPIAKNNATELVLAGDDAQLIYDERGANYKLEHKLIERSEIILNLTETRREGSEIANLATAIIDDMNYIQKREVLSAATHSTKVAHIDLFQ1vEvISILQMATTDLTVYILARTNAVLNYVAKVLDEYKIQYKKNERITDFDRELLSLNRLMRNEYTNDDIYITYNYLRNKVAREEELKERLFQHKLHWTEKDVLGILLLAYEQTTAKRILTTAKNINFKIKLSTIHSAKGSEADVVFLINSVPHKTKMKILENYEGEKRVLYVAVIRARKFLFIVDQPVARRYEQLYYIRSYESRAQGSLVNRVAVPVA. >tr|Q5JFK3|Q5JFK3_THEKO DNA helicase, UvrD/REPfamilyOS = Thermococcus kodakarensis (strain ATCC BAA-918/JON12380/ KOD1) GN = 1K0178 PE = 4 SV = 1 (SEQ ID NO: 64)MNEKEVILSKFIAHLKELVEMERRAEIEAMRLEMRRLSGREREKVGRAVIGLNGKVIGEELGYELVRYGRDREIKTEISVGDLVVISKRDPLKSDLVGIVVEKGKRFLIVAIETVPEWAIKGVRIDLYANDITEKRWMENLDNLRESGRKALELYLGLREPEESEPVEFQPFDKSLNASQRGAIAKALGSGDFFLVHGPFGTGKTRILVELIRQEVARGHKVLATAESNVAVDNIVERLADSGLKVVRIGHPSRVSKALHETTLAYLITQHDLYAELRELRVIGENLKEKRDIFTKPAPKYRRGLSDREILRLAEKGIGVPARLIREMAEWIRINQQVQKTEDDARKLEERIAREIIQEADVVLITNASAGLEVVDYGEYDVAWIDEATQATIPSVIIPINRAKRFVLAGDHKQLPPTILSEKAKELSKTLFEGLIERYPEKSEMLTVQYRMNERLMEEPSREFYDGKIKARESVKNITLADLGVSEPEFGNEWDEALKPENVLVFIDISKREDRFERQRRGSDSRENPLEAKLVTETVEKLLEMGVKPDWIGVITPYDDQRDLISSMVGEDIEVKIVDGYQGREKETIVLSFVRSNRRGELGELTDLRRLNVSLTRAKRKLIAVGDSSTLSNHPTYRRFIEFVRERGIFIEIDGKKH >tr|C6A075|C6A075_THESMDNA helicase, UvrD/REPfamily OS =Thermcoccus sibaricus (strain MM739/DSM12597) GN = TSIB 2009 PE = 4SV =1 (SEQ ID NO: 65)MIRVQIPAGAPKYGPVAUGQSARLISGRSGVRSPAGPPKALLKERFRELFIHKNPVITMHVKNYIAKLVDLVELEREAEIEAMREEMRRLKGYEREKVGRAILNLNGKIIGEEFGFKLVKYGRKEAFKTEIGVGDLVVISKGNPLASDINGTVVEKGSRFIVVALETVPSWAFRNVRID LYANDITERRQLENLKKLSESGIRALKLILGKETPLKSSPEEFTPFDRNLNQSQKEAVSYALGEEDFFLIHGPFGTGKTRTLVELIVQEVKRGNKILATAESNVAVDNLVERLWGKVKLVRIGHPSRVSVHLKESTLAEWESHERYRKVRELRNKAERLAVMRDQYKKPIPQMRRGLINNQILKLAYRGRGSRGVPAKDIKQMAQWITLNEQIQKLYKFAEKIESEIIQEIIEDVDVVLSTNSSAALEFIKDAEFDVAIIDEASQATIPSVLIPIAKARREVLAGDHKQLPPTILSEEARALSETLFEKLIELYPFKAKMLEIQYRMNQLLMEFPSREFYNGKIKADGSVKDITLADLKVREPFFGEPWDSILKREEPLIFVDTSNRIDKWERQRKGSTSRENPLEALLVREIVERLLRMGIKKEWIGTITPYDDQVDSIRSIIQDDEIEIHTVDGYQGREKEIIILSLVRSNKKGELGFLMDLRRLNVSITRAKRKLVVIGDSETLVNHETYKRLIHFVKKYGRYIELGDTGIN >tr|W0I5I1|W0I5I1_9EURY DNA helicase, UvrD/REPfamily proteinOS = Thermococcus paralvinellae GN = TES1_2001 PE = 4 SV = 1 (SEQ ID NO: 66)MNLIRYINHLKELVELEREAEIEAMREEMRKLIGYEREKVGRAVLGLNGKIIGEEFGYKLVKYGRKQEIKTEISVGDLVVISKGNPLASDLIGTVTEKGKRFLVVALETVPSWALRNVRIDLYANDITFKRQTENLDKLSESGKRALRFILGLEKPKESIDIEFKPFDEQLNESQKKAVGLALGSEDFFLIHGPFGTGKTRIVAEVILQEVKRGKKVLNFAESNVAVDNLVERLWGKVKLVRLGHPSRVSKHLKESTLAYQVEIHEKYKRVREFRNKAERLAMLRDQTKPITQWRRGLIDRQILRLAEKGIGARGIPARVIKSMAQWITFNEKVQRLYNEAKKIEEEIVKEIIRQADVVLSTNSSAALEFIKDIKFDVAVIDEASQATIPSVLIPIAKANKFILAGDHKQLPPTILSEEAKELSETLFEKLIELYPSKAKMLEIWRMNERLMEFPSKEPLINGKIKAYDGVKNITLLDLGVRVFSFGEPWDSILNLKEPLVFVDTSKHPEKWERQRKGSLSRENDLEAELVKEIVQKLLRMGIKPESIGVITPYDDQRDLISLLLENDEIEVKIVDGYQGREKEVIILSFVIRSNKKGELGFLTDLRRLNVSLTRAKRKLIAIGDSETLSAHPTYKRFVEFVKEKGIFVQLNQYVSQTS >tr|B7AA42|B7AA42_THEAQ DNA helicase OS =Thermus aquaticus Y51M023 GN = TaqDRAFT_3809 PE = 4 SV =1 (SEQ ID NO: 67)MGEAHPSEEALLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVERVAYLIARRGVEPSEILAVTFINKAAEEMKARLKAMVRGAGELWVSTFHAAALRILRVYGERVGLKPGFVVYDEDDQTALLKEVLKELGLAAKPGPIKSLLDRAKNQGVPPEHLLLELPEFYAGLSRGRLUVLHRYQEALRAQGALDFGDILLYALKLLEEDGEVLKRVRKRARFIHVDEYQDINPVQYRFIRLLAGEEANLMAVGDPDQGIYSFRADIRNILDFTQDYPKARVYRLEDNYRSTEAILRFANAVIVKNALRLEKTLRPVKKGGEPVRLFRAESARDEAREVAEEIARLGPPFDRVAVLYRTNAQSRLLEQALASRGIPARVVGGVGFFERAEVXDLLAYARLSLNPLDAYSLKRVLMTPPRGIGPATVEKVQAIARERGLPLFEALKVAALTLPRPEPLRAFLALMEELMDLAFGPARAFFRHLLLATDYPAYLKEAYPEDAEDRLENVEELLRAAKEAESLMDFLDKVALTARAEEPAEAEGRVALMTLHNAKGLEFPVVELVGVEEGLLPHRSSLSTQEGLEEERRLFYVGVTRAGERLYLSYAQEREIYGRLEPVRPSRFLEEVDEGLYEVYDPYRQSSRKPIPPPHRALPGAFRGGEKVVEPREGPGTVAAAGDEVIVHFEGVGLKRLSLKYADLRPA >tr|B7A5161B7A516 THEAQ DNA helicase OS =Thermus aquaticus Y51MC23 GN = TaqDRAFT_5093 PE = 4 SV =1 (SEQ ID NO: 68)MRVYLASAGTGKTHALVEELKGLIQSGVPLRRIAALTFIRKAEELRGRAKRAVLALSAEDPRLKEAEREVEIGALFTTIEIGEMATEALRHTAPLLSLDPDFALLDTFLAEALFLEEARSLLYRKGLDGGLARALLHLYRKRTLAETLHPLPGAEGVFALYLEALEGYRRRLPAELSPSDLEALALRILENPEALRRVVEREPHILLDEYQDTGPLQGRFFQGLKEAGARLVVVGDPKQSIYLERNARVEVEREALKQAEEVRYLSTTYRHAQAVAEFLNRETALFGEEGVRVRPHRQEVGRVEVHWVVGEGGLEEKRRAEAHLLLDRLMALREEGYAFSQMAVLVRSRSSLPPLEAAFRARGVPYALGRGRSFFARPEVRDLYHALRLSLLEGPPGPEERLAILAFLRGPWVGLDLSEVEEALKAQDPIPLLPEAVRAKLRALRALAGLPPLEALKRLSRDEAFLRRLSPRARVNLDALLLLAAMERFETLEALLEWLRLRAEDPEAAELPEGEEGWVLIVHGAKGLEWPVVALFDLSRGENPKEEDLINGLGGEVALRGTPAYKEVRKALRKAQAEEARRLLYVALSRARDVLIVTGSASGRPGPWVEALERLGLGPESQDPLVRRHPFKALPPLGDRPQTPPPPPLPAPYAHLAFPERPLPFVYSPSAFTKAKEPVPLAEALEKEALPEFYRALGILVHYAIARHLDPEDEGAMAGLLLQEVAFPFAEGEKRRLLEEVRDLLRRYRGMLGPSLPPLEAREEDHAELPLVLPLGGTVWYGILDRIYRVGGRWYLEDYKTDREVRPFAYRFQLAIYRRALLEAWGVFAEARLVYLRHGLVHPLDPEELERALKEGFPGMGPGEGGEKA >tr|B7A954|B7A954_THEAQ_DNA helicase OS =Thermus aquaticus Y51MC23 GN = TaqDRAFT_4764 PE = 4 SV =1 (SEQ ID NO: 69)MKGLIGSSRLRVYGPPGTGKTTWLKNEVERLLRSGVPGEEIAVCAFSRAAFREFASRLAGQVPEENLGTIHSLAYRAIGRPPLALTKDALSDWNRRVPDTWRVTPRVDGRGADLLDVMDPYEDEDSRPPGDKLYDRVAYLRNTLAPMAAWSEEERAFFQAWKSWMNAKGLVDFPGMLEAALAKPGGLGARFLLVDEAULTPLQLLLVEKWAQGARLALVGDDDQAIYGFMGADGASFLGVPVEDELVLGQSYRVPARVQRVAEAVIRRVQNRAPKRYAPRGDEGEVRLLWVPPEDPYHAVVDALERVNRGESVLFLATAKYLLEELKRELLRVGEPYANPYAPHRHSFNLFPQGARSAWEKARSELFPNRIAADVKAWTKHVSSKVEAVKGEEARRYLESFPDEEKVGDDHPIWNVERPEHRPHAVGRDVSWLLDHLLGNAPKTMRQSLMVAIKSPEAVLQGRARVWIGTIHSVKGGEADWVYVWPGYTRKAAREHPDQLHRLFYVAAJTRARKGLVLMDQGKAPHGYVWPRVDEFWGEVWV >tr|H7GEQ71H7GEQ7_9DEINDNA helicase OS = Thermus sp. RL GN =RLTM0_2916 PE = 4 SV = 1 (SEQ ID NO: 70)MEANLYVAGAGIGKTYTLAERYLGFLEEGLSPLQVVAVTFTERAALELRHRVRQMVGERSLGHKERVLAELEAAPIGTLHALAARVCREFPEFAGVPADFQVMEDLEAALLLEAWLEEALLEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRXLRRRMEELFHGERPEERYPGFPKGWRXEEPEVVPDLLAWAGEVKFNKKPWLEYKXDPALXRLLKLLGGVKEGFSPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEXEEVRAYYRGRERRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADPKVMERFQXEAAKEGLRVRRLEKSHRYHQGLADFHNRFFPPLLPGYGAVSAERKPEGEGPWVEHFQGDLEAQARFIAQEVGRILSEGFQVYDLGEKAYRPMSLRDVAVLGRTWRDLARVAEALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPXDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGILDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVTLLTVHGAKGIEWPVVEVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEXGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAARDPEAARRRLLGEPEXEGGEAWXPXXPQETEEEVPGGAGVGRMTHALLERFEAXEDLEREGRAFLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAXAYLLQVGVYALALGKPRALVADLREGKLYEGASQWEEKAEEVLRRLMGGEGQGRQPYPLAATDPGHGAPG >tr|H7GE69|H7GH69_9DEINDNA helicase OS = Thermus sp. RL GN =RLTM07977 PE = 4 SV = 1 (SEQ ID NO: 71)MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVTFINKAAEEMRERLRGLVPGAGEVWVSTFRAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAYGDPDQGIYSFRAADIKNILDFIRDYPEARVYRLEENYRSTEAILRXANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGEYERAEVKDLLAYARLALNPLDAVSLKRVINTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFXRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVELVGVEEGLLPHRNSLSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRXPKPXPPPHRPRPGAFRGGERVVHPREGPGTVVAAQGDEVIVHFEGXGLKRLSLKYAELXPA >tr|A0A0B0OSAG4|A0A0B0SAG4_9DEINDNA. helicase OS =Thermus sp. 2.9 GN = Q117 08170 PE = 4 SV = 1 (SEQ ID NO: 72)MDEALLSSLNEAQRQAVLHFQGPALVVAGAGSGKIRTVVHRVAYLIAHRGVYPTEILAVTFINKAAEEMRERLKGMVRGAGEVWVSTFHAAALRILRVITGERVGLKPGFVVYDEDDQTALLKEVLKELGLSAKPGPIKALLDRAKNRGEPPEALLAELPEYYAGLSRRRLLDVFERYQEALKAQGALDEGDILLYALRLLEEDQEVLARVRKRARFIHVDEYQDTNPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILQFLADFPGAKVYRLEENYRSTEAILRFANAVIVKNALRLEKTLRPVKRGGEPVRLFRAKDAREEAREVAEEILRLGPPFDRIAVLYRTNAQSRLLEQALAGRGVGARVVGGVGFFERAEVKDLLAYARLALNPLDSVSLKRILNIPPRGIGPATVEKVARLAQEKGLPLFEALKRAELLPRPEPVRHEVALMEELMDLAFGPAEAFFRHLLQATDYPAYLREAYPEDHEDRLENVEELLRAAKEAESLLDFLDKVALTARAEEPAGAEGKVELMTLHNAKGLEFPVVELVGVEEGLLPHRNSLITTLEALEEERRLFYVGV7RAQERLYLSYAEEREVIGRLEATRPSRFLEEVEEGLYQEYDPYRSPRPVPPSHRPKPGAFKGGEKVVEPREGPGTVVAASGDEVIVHFEGVGLKRLSLKYADLRPA >tr|A0A084IL47|A0A084IL47_9GAMMATP-dependent DNA helicase RepOS = Salinisphaera hydrothermalis C41138 GN = rep PE = 3 SV = 1 (SEQ ID NO: 73)MALPKLNPQQDAAMRYLDGPLLVLAGAGSGKTGVITRKIAHLIARGYDARRVVAVTFTNIKAAREMKQRASKLISADDARGLIVSTFESLGLQMIREEHAALGYKPRESIEDSEDADKVLADLVGRDGDHRKATKAAISNWKSALIDPETATAQATGSDIPLARAYGEYQRRLKAYNAVDFDDLLALPVHLLSTDHEARERWQSRFRYLLVDEYQDTNAAQYEMMRLLAGARAAFTVVGDDDQSIYAWRGARPGNIADLSRDEPHLKVIKLEQNYRSVGNVLSAANQLIGASNQRAYEKTLWEAMGPGDRVRVIAAPDEAGEAERIASEISSHKLRLGTAYGDYAILYRGNEQSRAFEKALRERDIPYRVSGGRSFFERSEIRDLVTYLKLMVNPDDDAAFLRIVNIPRREIGPATLEALGRYAGSRHISLFDAARGIGLAGGVGERSGRRLADEVDWLRNLTUSEGMTPRELVSQLIVDIDYRNWLRDTSANTKAARKRIENLDDFIGWLDRLDNAEDGKPVTLEDVVRRISLMDFANQSEKDVENQVHLLTLHAAKGLEFDHVFLAGLEEGMLPHHACLEDDKIEEERRLLYVGITRARKTLALTYARKRRRGGEESDSVPSRFLEELPADELDWPSATGTRSKAANAEQGRDQVAALRAMLGASADS >tr|A0A0A2WMV1|A0A0A2WMV1_THEFIDNA helicase OS = Thermusfiliformis GN = THFILI00990 PE = 4 SV = 1 (SEQ ID NO: 74)MPQVGFTDHEFKGLEALSREEQNRVREAVFAFMQDPKHPSFKLHRIEDIKTDREWSARVSKDLRLILYHHPEMGWIFAYVGHHDDAYRWAETHQAEVHPKLGLLQIFRVVEEVRVEPRKIKPLLPDYPDDYLLDLGVPPSYLKPLRLVETEDQLLGLIEGLPQDVQERLLDLAAGRPVTLPPKLAPSEETNEKHPLSRQHIHFIQNLDELRQALSYPWERWMVELHPAQREAVERVFQGPARVTGPAGTGRTVVALHRAAALARRYPEEPLLLTTENRFLASRLRSGLQRLLGEVPPNITVENLHSLARRLHEQHVGPVIKLVKEEDYGPWLLEAAQGLEYGKNFLLSEFAFADAWGLYTWEAYRGFPRTGRGVPLTARERLKLFGAFQKVWGRMENEGALTENGLLHRLRQRAEEGALPRFRAVVVDEAQDLGPAELLLVRALAQEAPDSLFFAIDPARIYKSPLSWQAIGLEVRGRSIRLKVNYRTTREIAKRAEAVLPKEVEGEMREVLSLLQGPEPEIRGFPTQEACQAELVRWLRWLLEQGVRPEEVAVLARVRKLAEGLAEGLRRAGIPVVLLSDQEDPGEGVRLGTVHSAKGLEFRAVALFaANRGLFPLESLLREAPSEADREALLAQERNLLYVAMSRARERLWVGYWDEGSPFLTP >tr|A0A0D0N7B7|A0A0D0N7B7_MEIRU DNAhelicaseOS = Meiothermusruber GN = SY28 04645 PE = 4 SV = 1 (SEQ ID NO: 75)MSDLLSSLNPSQREAVLHFEGPALVVAGAGSGKTRYVVHRIAYLLRERRVYPAEILAVIFTNKAAGEMKERLEKMVGRSARDLWVTTFHAAAVRILRTYGEYVGLKPGEVIYDEDDQNTLLKEVLKELELEAKPGPFRSMIDRIKNRGAGLAEYMREAPDFIGGVPRDVAAEVYRRYQNSLRMQGALDENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVORLTRLLAGERPNLMVVGDPDQSIYGFRNADINNILDFIKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTKPGGEPVRLYRAPNAREEAAFVAREIVKLGGYQQVAVLYRTNAQSRILEEHLRRANVPVRLVGAVGFFERREIKDLLAYGRVAVNPDDSINLRRIVNTPPRGIGATTVARLVEHAQKTGITVFEAFRAAEQVISRPQQVQAFVRLLDELMEAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRLAQDWEEEEGGSLADFLDSVALTAKAEEPQGDAPVEAVTLMTLHNAKGLEFPTVYLVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERETYGKREYTRPSRFLQDIPQDLLKEVGAFGDGETRVLSQARPEPKPRTQPAEFKGGEKVKHPKFGSGTVVAAMGGEVIVMFPGVGLKRLAVKFAGLERLE >tr|W2U4X3|W2U4X3_9DEINDNA helicase OS =Thermus sp. N1VIX2.A1 GN = TNMX_07060 PE = 4 SV = 1(SEQIDNO:76)MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRIV-VHRVAYLIAKRGVEPSEILAVTFINKAALEMRERLKRMVKGAGELWVSTEHSAALRILRVYGERVGLKPGFVVYDEDDQTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRILDVLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDINPVQYRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFTRDFPGAKVYRLEENYRSTEAILRFANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEAREVAEEILRLGPPFDRVAVLYRTNAQSRLLEQALASRGVPARVVGGVGFFERAEVXDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALAREKGLPLFEALRVAAEVLPRPAPLRHFLALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPEDHEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLSTLEGLEFERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSPAPSEARASKPKPGAYRGGEKVIHPREGQGTVVAAMGDEVTVHFEGVGLKRLSLKYADLRPVG >tr|H9ZQB5|H9ZQB5_THETHDNA helicase OS =Thermus thermophilus IL-18 GN = TtJL18 0620 PE = 4 SV =1 (SEQ ID NO: 77)MSDALLAPLNEAQRQAVLHFEGPALVVAGAGS GKTRTVVERVAYLVARRGVEPSEILAVTFINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLEALLGELPEYYAGLSRGRLADVLVRYQEALKAQGALDFGDILLYAIRLLKEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFIRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRLANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVIYRTNAQSRLLEQALAGRGIPARVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGIPPWEALKEAARTSSRVEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYLREAYPEDAEDRIENVEELLRAAKEAEDLQDFLDKVAITAKAEEPAEAEGKVAIMTLHNAKGLEFPVVFLVGVEEGLLPHRNSLSLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREARESRFLEEVEELYEVYDPYRVPKPAPPPHRPRPGAFRGGERVVHPRFGPGTVVAAQGDEVTVHFEGFGLKRLSLKYAELRPA >tr|E8PM35|E8PM35_TRESS DNA helicase OS =Thermus scotoductus (strain. ATCC 700910/SA-01) GN = perAl PE = 4 SV =1 (SEQ ID NO: 78)MQGPQSSHPGDELLRSLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVHRVAYLIAKRGVFPSEILAVTFTNKAAEEMRERLKRMVKGGGELWVSTFHSAALRILRVYGERVGLKPGFVVYDEDDQTALIKEVLKELGLAARPGPLKALLDRAKNRGEAPESLLSELPDYYAGLSRGRLLDVLKRYEEALKAQGALDFGDILLYALRLLEEDPEVLKRVRRRARFIHVDEYQDTNPVQRFTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILEFIRDFPGAKVYRLEENYRSTEATLRFANALIVNNALRLEKTLRPVKPGGEPVRLYRARDARDEARFVAEEILRLGPPFDRVAVLYRTNAQSRLLEQTLASRGVPARVVGGVGFFERAEVKDLLAYARLSLNPLDGVSLKRVLNTPPRGIGPATVEKVEALARKGLPLFEALRVAAEVLPRPAPLRHFLALMEELQELAFGPAEGFFRHLLEATDYPAYLREAYPEDYEDRLENVEELLRAAKEAEGLMEFLDKVALTARAEEPGEPAGKVALMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRTEATRPSRFLEEVEGGLYEEYDPYRASAKVSPSPAPGEARASKPGAYRGGEKVIHPREGQGTVVAAMGDEVIVHFEGVGLKRLSLKYADLRPVG >tr|E8PL08|E8PL08_TRESS DNA helicase OS =Thermus scotoductus (strain ATCC 700910/SA-01) GN =perA2 PE = 4 SV =1 (SEQ ID NO: 79)MLNPEQEAVANHFTGPALVIAGPGGKTRTVVHRIARLIRKGVDPETVTAV7FTKKAAGEMRERLVHLVGEETATKVFTATFHSLAYHVLKDIGTVRVLPAEQARKLIGEILEDLQAPKKLTAKVAQGAFSRVKNSGGGRRELIALYTDFSPYIERAWDAYEAYKEEKRLLDFDDLLHQAVHELSTDIDLQARWQHRARFLIVDEYQDINLVQFNLLRLLLTPEENLMAVGDPNQAIYAWRGADFRLILEFKKHFPNATVYKLHTNYRSHNGIVTAAKKVITHNTQREDLDLKALRNGDLPTLVQAQSREDEALAVAEVVKRHLDQGTPPEEIAILLRSLAYSRPIEATLRRYRIPYTIVGGLSFWNRKEVQLYLHLLQAASGNPESTVEVLASLVPGMGPKKARKALETGKYPKEAEEALQLLQDLRAYTGERGEHLASAVQNTLHRHRKTLWPYLLELADGIEEAAWDRWANLEEAVSTLFAFAHHTPEGDLDTYLADILLQEEDPEDSGDGVKIMTLHASKGLEFAVVLLPFLVEGA.FPSWRSAQNPATLEEERRLFYVGLTRAKEHAYLSYHLVGERGATSPSRFARETPANLIHYNPTIGYQGKETDTLSKLAELF >tr|E4U8J8|E4U8J8 OCEP5 DNA helicase OS =Oceanithermus profundus (strain DSM14977/NBRC 100410/VKMB-2274/506) GN =Ocepr 1221 PE = 4 SV = 1 (SEQ ID NO: 80)MSARDLLSSLNEQQQAAVQHFLGPAIVIAGAGSGKTR7VVHRVAYLLAEREVYPAEVLAVTFINKLAGEMRERLSRMVGRAAGELWVSTFHSASLRILRRYGERIGLKPGFVVYDDDDQRVLLKEVLGSLGLEARPTYVRAVLDRIKNRMWSVDEFLAHADDWVGGLIKQQMAEVYARY0QRLAENNAVDFNDLLLRTIELFERHPEALEAVRQRARFIHVDEYQDINPAQYRLTKLLAGDEANLMVVGDPDQSIYGFRNADIQNILGFERDYRGAVVYRLEANYRSTAAILRVANALIERNQQRLEKTLRPVKPAGEPVRLYRAPDHREEAAFVAREVARLAGERALDDFAVIYRTNAQSRVLEEAFRRLNLPARIVGGVGEYERREVKDVLAYARLAVNPADDVALRRVTNVPARGVGAASVGKLAAWAQAQGVSLLEAAHRAGELLAARQAAAVAKFTDLLTTLREAAEGTGPEAFLRLVLAETGYSEMLRREGDSEPRLENLEELLRAAEWEEEHGGSVAEFLDEIALTARAEEPNAAPEKSVILMTLHNAKGLEFPVVFVVGVEEGLLPHRSSLGSDAEIEEERRLLYVGITRAQERLYLTLSEERETWGQRERVRPSRFLEEIPEDFLKPVGPFGDAHEPAPAPLSSAPVNRAAKGSASGERGGEKVRHPRYGEGTVVATSGEGARQEVIVHFAEAGLKRLLVKYAGLERIE >tr|E4U4N5|E4U4N5_OCEP5 DNA. helicase OS =Oceanithermus profundus (strain DSM14977/NERC 100410/VKMB-2274/506) GN =Ocepr 1575 PE = 4 SV = 1 (SEQ ID NO: 81)MKVRIASAGTGKTYALTSRFTAALAEHPPYRLAAVTFTRSAAAELKARLRERLLAIAAGRFUSGAEDVPPEAVVRRAGALATEVLGATVTTIHGEFAELLRQNALALGLEPDFLRIDASESQQIFAEEARAYVYLNEEDDALAEVLGRLFAKRSLAAELRPQGEAAEALWAHFRAVLERYARRLGGEALGPADIELHAWRILERAGREEALAARIRSRLARVEVDEYQDTSPLQGRVFAALEALGVEVEVVGDPKQSIYAFRNADVEVEREAMRRGEPLPPLVTSWRHDRALVRFLNRYVDWVAEERPEAFARAEAPPVEARPDAGPGRVRLQLVQGEARQDALRPYEADQLARWLQERHAEHAWRDMAVLVRSHSSVPLLVRALAAHGLPHVVVGGRGEYDLIEVRDLVHAARVALDPRGRFSLAAFLRGPFAGLDLGRVERVLAAEDPLAELERAPEVAERVDRLVRWVQLRPLDFFERMVRTPFLEGASYLERLEPPARANVDQLLFKLASRRYGRLEFLLRDLEDLRGSDEAGVPEGGFDAVRIYTMHGSKGLETNPVVAVEDLNRGUDGAEPFYVRPGSGEFAAEGDPDYPRFAAEWKERERQEAYRILYVALSRPRSRILLSLSVQLKPDGEGLRPKEWRRILGRTLIEEMNLAAWDALEVERLDAARLPAPKAPRRAADVDERLRAPVEPLARPPVYSPSALKAERPAPPELDDEGDVAVELEEPGVDPGLVARTVGILVHYAIGQDWGPERLQDLWNQEAVQRLTEPERTRVKTEVAQRLETYWRLLGTELPALDERDEDYAEFPLLLPIRTARLDTVWEGVIDRLYRVGDVWVLEDYKTDRELHPERYHFQLALYRRAVAAAWGIEPEARLVYLRFGEVVPLEAQLLEEAFERGTREAEEV >tr|E4TJAI1|E4UAI1_OCEP5 DNA heiicase OS =Oceanithermus profundus (strain DSM14977/NBRC 100410/VIMB-2274/506) GN =Ocepr_2312 PE = 4 SV = 1 (SEQ ID NO: 82) MKVIVASA =KTTRLTQRYLEHLEQHPPQRVAAVTFTNKAAAELRERIFEALGRGSFYDFTPSPALAERLADYQVRVLEAPIGTIHSFFGYLLRLTAPMLGLDPHFEVTDPATARAWFLEEVRNLAIIEGAEVDETVITALVELFKRRSISEAFEGTGDASRSLVAGFKKVYARNLTRLGGRYLDPSEIERRALALIRHPEALERVRSRLGVVLVDEYQDTAPIQARVFEALEEAGVPIEVVGDPKQSIYAFRDADVEGFREAERRARENGNVETLIVSYRHPPALADFLNAFTSAEAALGKAFTAEEAPEVKPGREGDARVELITVIPGDGKATLDALRNGEARLLARELRRLHDEEGYDYGQMLVLERRRHQLPPLLRALRGAGLPFAVVGLRGLYEEPEVRELYHALRLATGEAPRDSLAVELSGPFGGLTLGQVREVLAQDAPESYLTLHEPEAAERLLRLRADAEKMRPAEALTRLIEAPTAKGPPFLDLLELEMADTVLYVLGRIEHTRTYPEAVATLESFRSGGEEEASLARLGGDAVRVMSAHAAKGLQAPVVVIFDADRTFNGNSDELVIEPRIGRVALNGEDAYESIAQALKARKEGEDHRLIYVALSRSSERLIVSAAVKEPRKGSWLHHLTEVLNLGSKFEHRNVTLAEIALEEPIEQEAATLPVDPELATPLPPAPPAVSSPTALKAERELEVPDPEEAWPADPEARLLGRIVGILVHEGIQRDWDPDDPEVLLALEGEQVLEEVPADRRPAVIEEVAILLRVYRTLLGSAIPSLEEREVDLAELPLVYPLGATAWEGVIDRLYRVGDVWYLEDYKTDREVHPERYHSQLALYREAVRKHWGIEPEVRLVYLRTGQVVPLDAAALKEGLASYTGG >tr|E4UAI8|E4UAI8_CEPS DNA helicase OS =Oceanithermus profundus (strain DSM14977/NBRC 100410/VIMB-2274/506) GN =Ocepr 2319 PE = 4 SV = 1 (SEGID NO: 83)MNEHERVIAHEVGPAAVVAGAGSGKTRAATLRAARLARTGERVGLVTFTASAAEEMRQRVLAEDVPAKHVWAGTFHSLAFQILRQFPEAGGYEGFPEVLTPNDELRLFRRLWAELLDQDLDAELRRKLVKALGFFRKARAEEALEGWAARAGESLELDAEMLEALMISFQLRKREAGLASFDDLIEGASRALGDKDVRKWADRRFPFLIVDEYQDTSRAQETFLAALMPGEAPNLMVIGDPNQAIYGWRGAGSRTFERFQARYPQAVLYPLRKNYRSTRAVLRIAERAIARIYRSGUAYYRLEGVKEEGEPPVLLTPPNAAAEATDVAREVARAVASGVPPEEIAVLARSSMQLAGVEDRLARLGVATRLLGGIRLSERREVKILVQLLKAAWSLHERALVDFIEEAVPGLGERTLTRVEHAARPYNLVDRIMNDGAFVRGFSTRVQQGLFMTRTLLQLARATFEGVTGEAFAERFREFAQDLYGELLPGYLARIGKQGPNEEARRRHLERFVATVEAFAREEAEGGLDDLLARLAFLEQQDGPAVTLGTVHAVKGLEFEVVFIVGMVEGAFPILADDSDPEEERRLFYVAAJTRAKRRLYLSAPTYGPRGKILUSRYLEEALDEGLVRLQKVRPAA. >tr|E4UAI4|E4UAI4_OCEP5 AAA ATPase OS =Oceanithermus profundus (strain DSM14977/NBRC 100410/VIB-2274/506) GN =Ocepr 2315 PE = 4 SV = 1 (SEQ ID NO: 84)MVSEGRWKIERVVYLKDGFAV-VAVRNEAGERHTAVGEMPTPVEGTWVRMETEHTVHPRYGPRLRVVRFLGLAPPPSKELAKIEGYLKLGFSEFLASWLAARFGSRPERAFDKPQELLVPGVPREVLRRVFPRLERLLGGLIDLLGEGHTAAPLFLLAERSGLGKEEIQELAREARKQRLIVEEQGRYGLVQPYRTERSIADGLLFRLKPGRGLRLIPPAGHGLSDEQARIFKLVRENRVVVLIGGPGSGKITTIATLLAAPELHRMREGIAAPTGKAARRIAEVARLPAETIHRLLGLGEARRPLYHARNPLPYDLLVIDETSMLDAEIAAFLVDALAPSTSWIFVGDPDQLPPVGPGQFLRDLMTRVATLRLTQIFRQAQDSPIVNGAYALREGRMPLADGERLRLLPFEEEAAQTTLRILLDELQRLEQIVGERPQNLVPGNRGPLGVRRLSPFLQQQLNPGGKPLGPIGWGMEAREGDPAVWIHNDYELGIMNGEVGVLRGGGSLGLTFETPTDRFAIPGNKRSRLVLAYAMTVHRSQGSEWPAVITILPKAHMALLSRELVYTALTRSKQYHTLLFHPEALYRARAVQASRRYTWLDVLLRG >tr|K7QW32|K7QW32_THEOS DNA helicase OS = Thermus oshimai JL-2GN = Theos_1787 PE = 4 SV = 1 (SEQ ID NO: 85)MTAPGHPDALLAPLNPAQQEAVLHFQGPALVVAGAGSGKTRIVVHRVAYLMAHRGVYPGEILAVTFTNKAAEEMKGRLKALVPGAGELWVATFHSAALRILRVYGEAIGLKPGFVVYDEADQEALLKEVLKELGLSAKPGPLKALLDRAKNRGEAWEALEIPDYYAGLPKGKVLDVLRRYQEALRAQGALDFGDILVYALRLLEENPEVLAKVRKRARFIHVDEYQDTSPVQYRFARLLAGEEANLMAVGDPDQGIYSFRAADIRNILDFTRDFPGARVIRLEENYRSTEAILRFANAY-1QKNRLRLEKTLRPVKPGGEPVRVIAAPEAREEARFVAEEIFRLGPPYERFAVLYRTNAQSRLLEQALAAKGIPYRVVGGVGFEERAEVKDLLAYARLSLNPEDGVSLKRVLNIPPRGIGPATLARIEALAQAEGVPLLGAIRLGAERFPKPEPLRAFLALLDFLADLAFGPPEAFFRELLSATDYLQYLKEHHPEDAEDRLENVEELLRAAKEAULQEFLDRVALTARADQDGGRGVALMTLHNAKGLEFPVVFLVGVEEGLLPHQSSLSTLEGLEEERRLFYVGVTRAQDRLYLSYAREREVYGRREPRRMSRFLEEVPEGLYLPHDPYRQGAUKPAPRAQGAFRGGEKVVHPREGPGTTVVAASGDEVIVHFEGVGLKRLSLKYADLRPA >tr|K7QWX5|K7QWX5 THEOS DNA helicase OS =Thermus oshimai JL-2 GN = Theos_2419 PE = 4 SV = 1 (SEGID NO: 86)MASSLSKAELVPTPEQEKALHLYRSRUFKLVAVAGSGKITTLRLMAESFPRRHIAYLAFNRAMKEEARRKFPPNTRVFTLHALAYRRTVPGTPYEAKFRLGNGQVRPVHVRERLQVDPLLAYVVRSGLERFIRSGDPEPLPRHLPRDWRKIVEARGPSGFAEVERAVKGVALLWKAMRDPHDPFPLSHDGYVRIWREEGAGGDPPAGWILVDEAQDLDPNELIVISGWRGKAQQVFVGDPRQQIYGWRGAVNAMGEIDLPESPLIWSFREGEPLASFVQAVTARQTQGLVPLVGRAGWATEVHVNLEPTPPFTILTRSNLGLVTALLEGAQLFSLQKEEAHVVGGVEFLVWLLTDLQAIKEGGERPRPHPELLGISKWEEVESLAEYSIVLNRLLRLAKEYDLEALAHKIAQLHGPEEGAKLVLSTAHKAKGREWDRVLLWEDFYWVAAYRWFFPNTAPPPSEPSPEFLEEENIFYVAMTRARLGLHISLPEALAEEEAKRILDRLSQGVPSGEDRGEDERGETLPAPFTGPTPVSPKEATFPLPSLYDRLLSEALNGGRDPLLHLLRDDLARLSALSPTPLPPEVAQALWERARPEEALGAIREGLGAMWREDPYELLRAINALALLGGRNPRKLAKILGDRFPGGEEAEDLLFVARARKRELMGRSLAEFWRGLGASVRHPLLKAYARAIRS >tr|K7QTS9|K7QTS9_THEOS DNA helicase OS =Thermus oshimai JL-2 GN = Theos_0356 PE = 4 SV = 1 (SEQ ID NO: 87)MRLYVASAGTGKTETLMGELKALLEGGVPLRRVAAVSFIRKSAEELRLRVRRLLEAHREAFWAREALREVEGALFTTLHGEMAEALRHTAPFLGLDPDFRVMDGFLAQALFLEEARSLLFLEGHPEAPELLELLEAGTGEKRSLAEAFTPLPGAEGLLALYERVLARYRARTQEVLGPGDLEAKALLLLRHPEALGRVAERFSHLLVDEFQDVNPLQGRFLRALEEAGVRVVAVGDPKQSIYLFRNARVEVFLRARAAEEVRALSRTHRHAKQVVELLNRETTREFRAEEGNRVEGVREAEGRVEVHWVLGKLEEARRAEARLLAQRLLALRAEGIPFGEMAVLVRARTSLPPLEKALRAAGVPFVRGRGQSFEARPEVRDLYHALRLALAERPYALEDRLSLLAFLRSPFLGLDLSELEEALRAEDPWPLLPKGVQEALEGLRALALLPPLEALRRLARDEGFLRRISRRARANIDILLLLAAGAREPTLEDLLLWLALRAKDPESVELPEGGGGVTLLTVHGAKGLEWPVVALYDVSRGPSERPPPLLVDEEGRVALKGTEAYRALLKEAERAEREEALRLLYVALSRARDLLLITGSTSQRPGPWAEALQALGLGPDAQDPWVETHPLEAIPPLPPIPQAPQDPRPAPYTPWRGEPRARPPVYSPSAHLKAEAEPLEVLGEGEALPEWARAVGTLVHYAIARHLDPEDEGAMGGLLRQEVALAFGEGEREALLEEVRALLRAYRSLLSGALPPLEARAEDHAELPLLLPHKGIVWYGVLDRLYRVGDRWYIDDYIKTDQKVRPEAYRFQLALYRKAVLEAWGVEAEARLVYLRHRQVVPLSPAELEAALEGL >tr|D1AF88|D1AF88_THECD DNA helicase OS = Thermomonosporacurvata (strain ATCC 19995/DSM43183/JCM3096/NCIMB 10081) GN =Tcur_4104 PE = 4 SV = 1 (SEQ ID NO: 88)MSSSQVTGRPTIVKDAEIAVEQRRVDQAHARLEEMRAEAQAMIEEGYRQALAGIKGSLVDRDAMVYQAALRVQALNVADDGLVEGRLDLADGQTRYIGRIGVRIRDHEPMWIDWRAPAAEAFYRATPEDPQGVVRRRVLHTRGRTVVDLEDDLLDPSAADSLTIVGDGAFIASLARTREGTMRDIVATIQREQDEVIRAPADGTVLVRGAPGIGKTAVALHRVAYLLFRERRREGSRGVLVVGPNRRETAYIERVLPSLGEGSATLRSLGDLVEGVSATVHDPPELAALKGSAAKAPVLRRAVTDHPPGAPDKLRVVHGGVVVELGRPQLDKLRTSLHRRSTGSVNASRRRVAEALLDALWERYVHIGGTEPEPDEPVQGELALWEGILAEGGLAPLDEQDRPSSPADRTSREAFVKNVREQRAFTDELTAWWPIRRPLDVLRSLGDAARLRRAAGRDLDRAQVELLAASWRRALAGDPPTLSYQDIALLDEIDALLGPPPQPSRATAREEDPYVVDGIDILTGEVVADEDWEPGLQELTTTIERLERARRVDDEVADVRPEYAHIVVDEAQDLSPNQWRMLGRRGRQATWTIVEDPAQSAWEDLEEARKAMEAALDGPAARRGRSRRPRRRPRHEYELTTNYRNTTEIAAVSARVLRLALPEARPARAVRSSGHRPVIDLVPEEELQAAARRAVRILLEQVEGTIGVIVPLPGDAWGESDRRALSAGFPERVQVLDVLEAKGLEFDAAVICAPETIAAQSPRGLRVLYVAVSRATQRLTVLTADPVWRRRLAGGESAR >tr|F8A884|F8A884 THEID DNA helicase OS =Thermodesulfatator indicus (strain. DSM15286/JON11887/0IR29812) GN =Thein 0607 PE = 4 SV = 1 (SEQ ID NO: 89)NTSISLDQYQEQAVKAKGNTLVVAGPGAGKIRVLLAKAIHLLEQGIDPEKVLILIFTIKTTQELKERLASIGIKGVKVDTFHALAYDLLKAKGIKPRLATEEELKALARDLSKRKGLSLKDERKALDKGENHYRSLWEEALKLHGLYDFSLLLKEATGHYLQQEKVYLLIDEFQDLNPELTSFLKTFTKAEFFLVGDPAQATYGERGACPQVIKEFVDYLAPQIYFLKKSYRVPEKVLNEAETLRETQGFPLEPLEAVQKGGNRLGLSENKPFNEAKGVAKLVSELLGGLQMEASQRGLAPPEIAILSRVRILLNPIKEAFIKEGIPFQVPSENLKEEISATESLSDIAKSIKSLKELEAYLAEGPSSVKEAWLESQSLEGFLFRLEMLKTFASISIRKDGVPLLTIHEAKGLEFKVVILVGAEDGLLPFTLLEDYDLAEEKRVAYVAVTRAQESFYFTWKIGRFLYGHKLSGKVSPFFETLPIKEKSSKTKPKARQKKLFG>tr|A0A087LEB0|A0A087LEB0_GEOSE Uncharacterized protein OS =Geobacillus stearothermophjius GN = GT94_17890 PE = 4 SV = 1 (SEQ ID NO: 90)NTISVIDELLEKNKQNNNKTAKDAVEAQLIAYAKKEVKKLUIRPHPYFGRLDFEDEFGRETIYIGKKGLEKDGELIVVDWRIDLGRLYNAYQGVQKTFQIGKENRPVTIHGKRGIVIKNGKVIKVIDIGKSEIIENDNGEKVKYNDDYLKEILTNTEEAHRLRDITASIQAEQDETIRLPLKDTIIVQGAAGSGKSTIALHRISYLLYWHEQVXPKDILILAPNEIFLSYIKDIVPEIEIEGIEQRTFYDWASTYFTDVHDIPDLHEQYVHIYGSTEKEDLIKIAKYKGSLRFKKLLDDEVEYIGNTNIPHGDVVIESGVILSKEEIHQFYHAKEHLPLNVRNKEVKEFIINWRNEQINIRKQQIEDEFEEAYRKWVVTLPEGEERKAVYEALEKAKQLRMKIFQEKNQHEISLIVKKMENIPALLNYKSVFQKKVFEKFHPDIDEELLSLLLKNGRQIKQERFMYEDIAPLIYLDAKINGKKLWEHIVIDEAQDYSPFQLAINKDYAKSMTILGDIAQGIFSFYGLDRWEEIESYVFKEKEFKRLHLQTSYRSTKQINDLANRVLLNSNIDEPLVIPVNRPGDVPTIKKVESIGELYDEIVNSIRIFEEKGYKKIAILTASKQGAIDTYDQLNRRQITQNEVITEGHQALKEKIVIIPSYLVKGLEFDAVIIEDVSDETEKDETQHAKMLYNSITRAHHDLHLFYRGNISPLLEERDPSAPPKPRKSFADWLITDINDPYVEPQVEAVKRVKKEDMIRLFDDEEEEFVEEAFEDDRERYYDFHAWLKVWRRWAEMRKQLDEKS >tr|B5Y6N2|B5Y6N2_COPPD DNA helicase OS =Coprothermobacter proteolyticus (strain ATCC 35245/DSM5265/DT) GN =perA PE = 4 SV = 1 (SEQ ID NO: 91)MALPQENLIPPSPSHNHLTLSLRSHIGGFFIYNEDVDSVDLSKLNEAQKQAVTAPPKPLAIIAGPGSGKTRVLTYRALFAVKEWHLPPERILAITFINKADELKERLGRLIPEGDRIFAATMHSFAARMLRYFAPYAGISQNFVIYDDDDSKGLIEDILKQMNFIDTKRFRPNDVLNHISAAKARMFDCNTFPEFIRQRYGSWGITYFDTVHQVFMTITERLKEQSQALDFDDLIMVLAQRNEDRPELRENTAGLFDLVMVDEFQDINFAQYQNLLYNTNPHYSGMNNVTIVGDPDQSIYGFRAAETYNIKRFIDDYNPEVVELDLNIRSNRTIVDSASALINDSPSALFERKLESIKGAGNKLILRRPFDDADAAITAAFEVQRLHKNGIPYEEIAVLMRTRALTARVEREFATRNIQYEIIGGVPFFARRETKDILAYLRLSRNAMDRVSLKR:ILTMKKRGFGTABLEKLENFAEENKLTLLEAMKAAVESLLFKKLSMNDYLESLYTLIQTIQEIAEPSQAIYLVNEUNLLDHFRSISKSEEEYIERTENVKQLISIAEESADMDDFLQRSALGTRENNGGVEGVAISTVHGVKGLEFQAVILYYVTDGFFPHSLSVITAEKEEERRLLYVAMTRAKEHLIFYVPYKQPWGNGFEQMARPSPFLRSIPKELWDGKPNEIESLYAPYBPQQKWSE >tr|D7BJL0|D7BJL0_MEISD DNA helicase OS =Neiothermus sjivanus (strain. ATCC 700542/DSM9946/VI-R2) GN =Mesil 3574 PE = 4 SV = 1 (SEQ ID NO: 92)MNDPIRHKEGPALVFAGAGAGKTRILTQRVKWLVEEGEDPYSITLVTFINKAAGEMKERIARLVEAPLAEAVWVGTFHRFCLQSLQVYGREIGLEKVAVLDSAAQRKLAERIIAGLFPAKPPRGFIPMAALGAVSRAANSGINDDIQLATMYADLTEKIVNERWAYEEAKKGLGALDYDDLLLRGVRLLKLSEGAARMVRRRAAYLMVDEFQDINGVQLELVRAIAPGISPNLMVVGDPDRSIYGWRGANYRTILEFRQHYPGAAVYGLYTNYRSQAGVVEVANRIIAQNATRKPEMQEAHLPQSEEPFLLVAKNRWEEAHFVAQAVEFYRGQGIALEENAVLNRANFLSRDLEQALRLRGIPYQFTGGRSFFERREIQLGMAVLKVLANPKDSLAVAALVEEMVEGAGPLGIQKVLEAAKAANLSPLEAFRNPAMVKGLRGKEVQAEANRLAEVLQDQVARLAAEAPEYHALLKEILDRLSTEAWIDRLGEESEQVYERKANLDRLLQGMQEWQEVNPGAPLQDLVGILLLEAGDIPAEEGQGVHLNIVHASKGMEFRVVEVIGLNEGLFPLSKASSSFEGLEEERRLNYVAV7RAKEVLHLSYAADGVVSRFAQEARVPVEEYDPRIGWSGRQNQQALKALLEIA >tr|E8MZN5|E8MZN5_ANATU DNA helicase OS =Anaerolinea thermophila (strain DSM14523/JON11388/NBRC 100420/UNI-1)GN = perA PE = 4 SV = 1 (SEQ ID NO: 93)MDSLEHLNPQQRAAVIASAGPVLVLAGPGSGKTRVITFRIGYLLSQLGVAPHHILAVIFTNKAARENQSRVEKLLGHSLQGMWLGTFHAICARdLRREQQYLPLDANEVIEDEDDQQALTKRALRDLNLDEKLYRPTSVHAAISNAKNNLILPEDYPTATYRDEVVARVYKRYQELLVSSNAVDFDDLLLYAWKLLNEFSTVREQYARRFEHILVDEFUTNLAQYELVKLLASYHRNLFVVGDEDQSIYRWRGADYRNVLRFEEDFPDRQKILLEQNYRSTQRVLDAAQAVINRNRNRIPKRLKSTPEHGEGEKLVLYEAVDDYGEAAFVVDTIQQLVAGGKARPGDFAIMYRTNAQSRLLEEAFLRAGVPYRLVGANRFYGRREVKDMIAYLRLVQNPADEASLGRWINVPPRGIGDKSQLALQMEAQRTGRSAGLILMELGREGKDSPHWQAIGRNAELLADEGSLLGEWHRIKDEISLPSLFQRILNDLAYREYTDDNTEEGQSRWENVQELLRIAYEYEEKGLTAFLENLALVSDQDTLPENVEAPTLLTLHAAKGLEFPIVFITGLDDGLIPHNRSLDDPEANAEERRLFYVGLTRAKKRVYLVRA_AQRSTYGSFQDSIPSRFLKDIPADLIQUGRGRRNGRSWQSESRRSWDDNYAGTWGSRPERAKPSHAPILQPRFKPGMRVKHPSWGEGLVVDSRIQDEDETVDIFFDSVGFKRVIASIANLEILS >tr|L0INW7|L0INW7_THEIRATP-dependent exoDNAse (ExonucleaseV), alpha subunit/helicase superfamily Imember OS =Thermoanaerobacterium thermosaccharolyticum M0795 GN = Thethe 02902 PE =4 SV = 1 (SEQ ID NO: 94)MDINGQIIKLNRNKTQGTLKLINGQKIKFKINSDSVKPIFLYEYYKFKGNMIEDTLIIDDIYGIANDININDFIELFPSVAHDKINNICNRENVLHVGNLIDLINDENFITVVNDTIGEEKATIFLSNLQKIKDRQEYIDVWDIIKKINPTEDINVPIKIVNALKYRASMNNITVSQLIKESPWIIEQLDIFDSITERKKIAENIATHYGLSNDSNKAVISYAIAMTNNYIQQGHSYIPYYILVSRVSNSLKLDENKVNDTLKFLPNDNKSGYLIRDNKYKDFIENFYNSDKKIGYSVYLPKIEHMEKYIADIISSILKKKSTINKIELQKNLKLYRSENKLIFSKEQEFAIFSISDNKITVITGGAGTGKITVIKAIIDLVNKMGYIPVVLAPTGIASQRVAPNVGSTIHKYARIFDTYDPVEDEIEENKENNSGKVIIVDEMSMITVPVFAKLLSVILDADSFIFVGDPNQLPPIGAGGVFEALIELGNKNINNINTVVLNQSFRSKNSIVXNAQNILEDKPIYEDDNLNIIEAKSWNKIADEVVNLIRKILDNGVQYSDIMVLSSKRGEGKNGVELLNERIRKEIENNKGKYAVGDIVITIRNDYDNKSSYFRSKELKKYINSIRHEERPTIENGTVGVIKDISDNEVIIEYNTPMPVEAKYNMEFLDWYIEYGFAITVHKAQGGQAKYIIFASDEPRNISREMLYTAITRCKNGKVFLIGGENEDWKIKKEHSFVLSKLKYRILDNIHQOEKESKINSKIVLINQ >tr|D3PR99|D3PR99_MEIRD DNA helicase OS =Meiothermus ruber (strain ATCC 35948/DSM1279/VKMB-1258/21) GN =K649 05745 PE = 4 SV = 1 (SEQ ID NO: 95)MSDLLSSLNPSQQEAVLHFEGPALVVAGAGSGKTRIVVHRIAYLLRERRVYPAEILAVTFTNKLAGEMKERLEKMVGRPARDLWVSTFHAAAVRILRTYGEYVGLRPGFVIYDEDDQNTLLKEVLKELELEAKPGPFRAMIDRIKNRGAGLAEYMREAPDFIGGVPKDAAAEVYRKYQSGLRMQGALDENDLLLLTIELFEQHPEVLHKVQQRARFIHVDEYQDTNPVQYKLTRLLAGERPNLMVVGDPDQSIYGERSADINNILDFIKDYPGARVIRLEENYRSSSSILRVANAVIEKNALRLEKVLRPTRPGGEPVRLYRAPNAREEAAFVAREIVKLGNEXIAVLYRINAQSRLLEFHLRRANVPVRIVGAVGFFERREIKDLLAYGRVAVNPADSINLRRIVNIPPRGIGATIVSRLVEHAQKTGTIVFEAFRVAEQVISRPQQVQAFVRLLDELIEAAFESGPTAFFQRVLEQTGFREALKQEPDGEDRLQNVEELLRPAQDWEEEEGGSLSDFLDSVALTAKAEEPQGDAPAEAVTLMILHNAKGLEFPTVFLVGLEENLLPHRNSLHRLEDLEEERRLFYVGITRAQERLYLSYAEERETYGKREYIRPSRFLEDIPQDLLKEVGAFGDSEVRVLPQARPEPKPRIQLAEFKGGEKVRHPKEGSGIVVAAMGGEVIVMFPGVGLKRLAVKFAGLERLE >tr|D3PLL2|D3PLL2_MEIRD DNA helicase OS =Meiothermus ruber (strain ATCC 35948/DSM1279/VIMB-1258/21) GN =K649 10770 PE = 4 SV = 1 (SEQ ID NO: 96)MKVRVASAGTGKIASLVLRYLELIAKGIPLRRIAGVTFTRKAADELRVRVAAAIEEVIQTGRELSEVASGGSRAAFQEAAREIAGATLSTIHGEMAQCLRLAAPILHLDPDFSMLGDWEAQAIFFEEWQTLRYLAQDAHHPLEGLVSDFLTEPLLHLFSRRSQAEVFEPAAGEANQHLLQVYQTVYAAYEARLGANLLSPSELERKALFLARNDRAMKRVLERVRVLLVDEYQDVNPVQGAFFLALEQARLPIEIVGDPKQSIYAFRNADVSVERKALREGKSEPPLIHSYRHSRVLVRFLNGLIGYLAKEGLGEGLEEAPPVEGVRPEQGRLEVHWVVGELPLEELRKQEARVLAGRLAALRGPIEYSQMAVLVRSYGSVRFLEEALAEAQIPYVLLQGRGYYERQEVRDLYHALRAALDPRGLSLAVFLRSPFGQHTEAGPLKPLELPQIEGVIRADDPLGRLAQHWPSVYERLRQIQAQVRLMAPLEVLKFLIRAPLMDGRPYHDFLEPRARENVDALLFYFAPRPPQNLEGLLERLELLSRQADAGDVPQSGEGVQILIVHQAKGLEWPLVAVEDLGRMNVHRPQPLYIGQGPNGGDGGRLRRWVALPETPQFEAFRQQVKLUEEESYRLLYVAASRARDILLLTASASHGQPEGWGKVLEAMNLGPASKPYHRPDFHLQIWPYQPAPPVRVLSQPAPLQPSPWVDARFEPEPFPPLFSPSALKRLEAEPLPLPDPEEGEAVPGRARAIGILVHYAIGQNWRPDNPQHLANLEAQEVMFPFGPDERRGIMAEVQALLEHYQELLGRALPWPRDEDYPEFAVALPLGSTVWQGVID RLYRVGQQWYLEDYKTDQEMRPERYLVQLGIYLAAIRQAWQIEPEVRLVYLRFGWVERLDKAILEAALGEIMPKGEGIRR >tr|Q9RTI9|0RTI9_DEIRA DNA helicase OS =Deinococcus radiodurans (strain ATCC 13939/DSM20539/JON16871LMG4051/NBRC 15346/NCIMB 9279/R1/VKMB-1422) GN = DR 1775 PE = 4 SV =1 (SEQ ID NO: 97)MTSSAGPDLLQALNPTQAQAADHFTGPALVIAGAGSGKIRTLIYRIAHLIGHYGVHPGEILAVTFINKAAAEMRERAGHLVPGAGDLWMSTEHSAGVRILRTYGEHIGLRRGEVIYDDDDQLDIIKEVMGSIPGIGAETURVIRGIIDRAKSNLWTPDDLDRSREPFISGLPRDAAAEAYRRYEVRKKGQNAIDEGDLITETVRLFKEVPGVLDKVQNKAKFITHVDEYQDTNRAQYELTRLLASRDRNLLVVGDPDQSIYKFRGADIQNILDFQKDYPDAKVYMLEHNYRSSARVLEAANKLIENNTERLDKILKPVKEAGQPVTFHRATDHRAEGDYVADWLTRLHGEGRAWSEMAILYRTNAQSRVIEESLRRVQIPARIVGGVGFYDRREIRDILAYARLALNPADDVALRRIIGRPRRGIGDTALQKLMEWARTHHTSVLTACANAAEQNILDRGAHKATEFAGLMEAMSEAADNYEPAAFLRFVMETSGYLDLLRQEGQEGQVRLENLEELVSAAEEWSQDEANVGGSIADFLDDAALLSSVDDMRTKAENKGAPEDAVTLMILHNAKGLEFPVVFIVGVEQGLLPSKGAIAEGPSGIEEERRIFYVGITRAMERILMTAAQNRMUGKINAAEDSAFLEDIEGLFDTVDPYGQPIEYRAKTWKQYRPTVRAATTAVKNTSPLTAELAYRGGEQVKHPKFGEGQVLAVAGVGERQEVTVHFASAGIKKLMVKFANLTKL >tr|M1E5C5|M1E5C5_9FIRM DNA helicase OS =Thermodesulfobium narugense DSM14796 GN = Thena 1375 PE = 4 SV =1 (SEQ ID NO: 98)MDLNLNEDQKRAVYSDSRALLIVAGAGTGKTRVLTTRAARLIKENPDARYLLLIFTKKAAREMTTRVRELIEEDTKNRLYSGTEHSFCSNIIRRRSERVGLINDFVIIDESDSLDLMKKVFSRIYSKEKIDSLIFKPKDILSLYSYARNNNQDFIEIVQRKYKYVNFEDIKKIISLYELNKKERNYLDFDDLLMYGLLAIKTLEKSPFDEVLVDEFUTNQIQAEMLYYFYDLGSRISAVGDDAQSIYSFRGAYYENMENYIKRLDAEKliLSSNIRSTQQILDIANSIIQSSYSSIKKELVANVRLKENVKPKLVIVSDDWEEARYVAREMQKFGEKGLKVAALYRAAYIGRNLESQLNSMGIKYSFYGGQKLTESAEAKDFMSFLRVEVNPKDEIALIRTLKMFPGIGEKKAFKIKDAVISGDNLKKALSKEKNLEELNIFFDKLFKITDWHDLLELVFDFYKDIMNRLYPENYEEREEDLIKEMDMSSNYDNLVEYLEAFTLDPVEKSEFDNNNVILSTIHSAKGLEFDVVFLLSVIESVYPHFRAQSTDEIEEERRLFYVAITRAKQRLIFTFPRHSKKSRGYFAKNTISPFLREKDNYLEVFIAR >tr|OSIE7|Q5SIE7_THET8 DNA helicase OS = Thermus thermophilus(strain. HB8/ATCC 27634/DSM579) GN = T1HA.1427 PE = 4 SV = 1 (SEQ ID NO: 99) MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVTFINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVITGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEAREVAFETARLGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFSRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRATIKEAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGLEFPVVELVGVEEGILPHRNSVETLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVEPREGPGTVVAAQGDEVIVEFEGFGLKRLSLKYAELKPA >tr|B5YD55|B5YD55_DICT6 DNA helicase OS =Dictyogiomus thermophilum (strain ATCC 35947/DSM3960/H-6-12) GN =DICTH_0581 PE = 4 SV = 1 (SEQ ID NO: 100)MNNQFDSEKKIFIIPSRKKKEFLERIEKDLNEEQRKVVLEADGPSLVIAGPGSGKTRTIVYRVGYIVALGYSPKNIMLLTFTNQAARHMINRTQALIRESIEEIWGGIFEHVGNRILRVYGKIIGINEQYNILDREDSLDLIDECLEELFPEENLGKGILGELFSYKVNIGKNWDEVLKIKAPQIIDKIEIVQKVFERYEKRKRELNVLDYDDLLFTWYRLLLESEKTRKILNDRELYILVDEYQDTNWLQGEIIRLTREENKNILVVGDDAQSIYSFRGATIENILSFPEIFPGTRIFyLVFNYRSTPEIINLANEIIKRNTRQYFKEIKPVLKSGSKPKLVWVRDDEEEAQFVVEVIKELHKEGVKYKDIGVLERSNYHSMAVQMELTLQGIPYEVRGGLRFFEQAHIKDMISLLKILFNEWEISAQRFFKLFPGIGRAYAKKLSQVLKESKDFDKIFQMUSGRTLEGLRILKNIWDKIKVIPVQNFSEILRVFFNEYYKDYLERNYPDFKDREKDVDQLILLSERYDDLEKFLSELTLYTYAGEKLLEEEEEEKDFVVLSTIHQAKGLEWHAVFILRLVQGDEPSYKSMDNIEEERRLFYVAVTRAKRELYVITYLTRKVKDMNVFTKPSIFLEELPYKELFEEWIVQREI >tr|F6DJA4|F6DJA4_THETG DNA helicase OS =Thermus thermophilus (strain SG0.5JP17-16) GN = Ththe16_124 PE = 4 SV =1  (SEQ ID NO: 101)MLSPFGGEEETKAIPLEFEILLAWRVFSAALPPNFLAPVSASLHILVREAEGKEGAELEAYAWERLEELARTSVVKDAIQSFLEVAAEKPEVLRAGLLWFRTWNRLSPEEREALYRKAERFKPTAELASKASFLQGPPPPPKPLSPSVQAARSSPPRFTPTPEQEEAVRAFLSREDMKLVAVAGSGKITTLRLMAQSAPKERLLYVAFNRSVRDEAERTFPGNVEVLTLHGLAHRHVVRGSGAYQRKLAARNGRVTPGDV-LEALELPRERYALAYVIRSTLEAFLRSASEVPIPAHIPPEYREVLQRRDKDPFSERYVLKAVRLIWKLMQDPDDSFPLSEDGFVKIWAQAGAKIRGYDAYLVDEAQDLSPVFLQNLEAERGELRRVYVGDPRQQTYGWRGAVNAMDKLDAPERKLIWSFRFGEDLARGVRRFLAHVGSPIELHGKAPWDTEVSLARPEPPYTALCRTNAGAVEAVISFLLEEGREGARVEVVGGVDEIANLLRDAHLLKVGGEREKPHPELALVENWEELEELAKEVNHPQARMLVRLARRYDLLELARLLKHAQADEEGKADLVVSTLHKAKGREWDRVVLWGDFIPVWDEKVREFYRKQGALDELKEEENVVYVALTRARRFLGLDQLPDLHERFFQGEGLVKPPSVSPLSVGGAGVSADLLRELEVRVLAKLEDRLKEVAEVLAALLVEEASKAVAEAMREMGLLGEEG >tr|F6DIL2|F6DIL2_THETG DNA helicase OS = Thermus thermophilus(strain SG0.5JP17-16) GN = Ththe16 1438 PE = 4 SV = 1 (SEQ ID NO:  102)MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRTVVERVAYLVARRGVEPSEILAVTFINKAAEEMRERLRGIVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFV-VIDEDDQTALLKEV-LKELALSARPGPIKALLDRAKNRGVGLKALLGELPETYAGLSRGRLGDVLVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFIRLLAGEEANLMAVGDPDQGIYSFRADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRLEKALRPVKRGGEPVRLYRAEDAREEARFVAEEIARLGPPWDRYAVLYRTNAOSRLLEQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNTPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFPRAEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPTYIREATPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGKVALMTLHNAKGLEFPVVELVGVEEGLLPHRNELSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVHPREGPGTVVAAQGDEVTVHFEGVGLKRLSLKYAELKPA >tr|F6DJ67|F6DJ67_THETG DNA helicase OS =Thermus thermophilus (strain SG0.5JP17-16) GN = Tnthe16 2078 PE = 4 SV =1(SEQ ID NO:  103)MEANLYVAGAGIGKTYTLAERYLGFLEEGLSPLQVVAVIFTERAALELRHRVRQMVGERSLGHKERVLAELEAAPIGTLHALAARVCREFPFEAGVPADFQVMEDLEAALLLEAWLEEALLEALQDPRYAPLVEAVGYEGLLDTLREVAKDPLAARELLEKGLGEVAKALRLEAWRALRRRMEELFHGERPEERYPGFPKGWRTEEPEVVPDLLAWAGEVKFNKKPWLEYKGDPALERLLKLLGGVKEGFSPGPADERLEEVWPLLRELAEGVLARLEERRFRARRLGYADLEVHALRALEREEVRAYYRGRFRRLLVDEFQDTNPVQVRLLQALFPDLRAWTVVGDPNQSIYSFRRADPKVMERTQAEAAKEGLRVRRLEKSHRYHQGLADEHNRFFPPLLPGYGAVSAERKPEGEGPWVEHFQGDLEAQARTIAQEVGRILSEGFQVYDLGEKAYRPMSLRDVAVLGRTWRDLARVAEALRRLEVPAVEAGGGNLLETRAFKDAYLALRFLGDPKDEEALVGLLRSPFFALTDGEVRRLAEARGEGETLWEVLEREGDLSAEAERARETLRGLLRRKALEAPSRLLQRLDGATGYTGVAARLPQGRRRVKDWEGILDLVRKLEVGSEDPFLVARHLRLLLRSGLSVERPPLEAGEAVTLLTVHGAKGLEWPVVEVLNVGGWNRLGSWKNNKTKPLFRPGLALVPPVLDEEGNPSALFHLAKRRVEEEEKQEENRLLYVAATRASERLYLLLSPDLSPDKGDLDPQTLIGAGSLEKGLEATEPERPWSGEEGEVEVLEERIQGLPLEALPVSLLPLAARDPEAARRRLLGEPEPEGGEAWEPDGPQETEEEVPGGAGVGRMTHALLERFEAPEDLEREGRAFLEESFPGAEGEEVEEALRLARTFLTAEVFAPYRGNAVAKEVPVALELLGVRLEGRADRVGEDWVLDYKTDRGVDAKAYLLQVGVYALALGKPRALVADLREGKLYEGASQWEEKAEEVLRRLMGGDRPEA >tr|G8N9P8|G8N9P8_9DEIN DNA helicase OS =Thermus sp. CCB US3 UF1 GN = TCCBUS3UF1 17030 PE = 4 SV =1 (SEQ ID NO: 104)MDAFPSGKPLDEAWLSSLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLMARRGVYPSEILAVTFTNKAEEMRERLKAMVKGAGELWVSTFHAAALRILRFYGERVGLKPGFVVYDEDDQTALLKEVLKELGVSAKPGPIKALLDRAKNRGEPPERLLADLPEYYAGLSRGRLLD-VLHRYQQALWAQGALDFGDILLLALKLLEEDPEVRKRVRKRARFIHVDEYQDTSPVQYRLTKLLAGEEANLMAVGDPDQGIYSFRAADIKNILUTEDFPGAKVIRLEENYRSTERILRFANAVIVKNALRLEKTLRPVKSGGEPVRLFRARDAREEAREVAEEVLRLGPPYDRVAVLYRTNAQSRILEQALASRGIGARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIPPRGIGPATVEKVQAIAQEKGLPLYEALKVAAQVLPRPEPLRHFLALMEELMDLAFGPAEAFFRHLLEATDYPAYLKEAYPEDLEDRLENVEELLRAREAEGLMDFLDKVALTARAEEPGEAGGKVALMTLHNAKGLEFPVVFLVGVEEGLLPHRSSVSTLEGLEEERRLFYVGVTRAQERLYLSYAEEREVYGRPEASRPSRFLEEVEEGLYEEYDPYRLPPPKPVPPPHRAKPGAFRGGEKVVHPRFGLGTVVAASGDEVIVHFDGVGLKRLSLKYADLRPA >tr|Q1J014|Q1J014_DEIGD DNA helicase OS =Deinococcus geothermalis (strain DSM11300) GN = Dgeo 0868 PE = 4 SV = 1 (SEQ ID NO: 105)MPDLPASSLLAQLNPNQAQAANHYTGPALVIAGAGSGKIRTLVYRIAHLIGHYGVDPGEILAVTFINKAAAEMRERARHLVEGADRLWMSTEESAGVRILRAYGEHIGLKRGEVIYDDDDQLDILKEIMGSIPGIGAETHPRVLRGILDRAKSNLLTPADLARHPEPFISGLPREVAAEAYRRYEARKKGQNAIDEGDLITETVRLFQEVPAVLERVQDRARFIHVDEYQDTNKAQYELTRILASRDRNLLVVGDPDQSIYRFRGADIQNILDFQKDYLDAKVYMLEQNYRSSARVLTIANKLIENNAERLEKTLRPVKEDGHPVLEHRATDQRAEGDEVAEWLIRLHAEGMRFSDMAVLYRTNAQSRVIEESLRRVQIPAKIVGGVGFYDRREIKDVLAYARLAINPDDDVALRRIIGRPKRGIGDTALERLMEWARVNGTSILTACAHAQELNILERGAQKAVEFAGLMHAMSEAADNDEPGPFLRYVIETSGYLDLLRQEGQEGQVRLENLEELVSAAEEWSRENEGTIGDFLDDAALLSSVDDMRTKQENKDVPEDAVTLMTLHNAKGLEFPVVFIVGTEEGLLPSKNALLEPGGIEEERRLFYVGITRAMERLFLTAAQNRMQYGKTLATEDSRFLEEIKGGFDTVDAYGQVIDDRPKSWKEYRPTESARPGAVKNTSPLTEGMAYRGGEKVRHPKFGEGQVLAVAGLGDRQEVTVHFPSAGTKKLLVKFANLTRA 7>tr|Q745W4|045W4_THET2 DNA helicase OS =Thermus thermophilus (strain HB27/ATCC BAA-163/DSM7039) GN =TT P0191 PE = 4 SV = 1 (SEQ ID NO: 106)MALRPTEEQLKAVEAYRSGQDLKVVAVAGSGKITTLRIMAEATPGKRGLYLAFNRSVQQEAARKFPRNVRPYTLHALAFRMAVARDEGYRAKFQAGKGHLPAQAVAEALGLRNPLLLHAVLGTLEAFLRSEAASPDPGMIPLAYRTLRAGIKTWPEEEAFVLRGVEALWRRMTDPKDPFPLPHGAYVKLWALSEPDLSFAEALLVDEAQDLDPIFLKVLEAHRGRVQRVYVGDPRQQIYGWRGAINAMDRLEAPEARLTWSFRFAETLARFVRNLTALQDRPVEVRGKAPWATRVDAALPRPPFTVLCRTNAGVVGAVVVTHEVHRGRVEVVGGVEELVHLLRDAALLKKGEKRTDPHPDLAMVETWEELEALAEAGYAPAYGVLRLAQEHPDLEALAAYLERAWTPVEVAAGVVVSTAEKAKGREWDRVVLWDDFYPWWEEGWRVNWGSDPAHLEEENLLYVAATRARKHLSLAOIRDLLEAVDRMGVYRVAEEATRAYLLLSAEVLRGVATDPRVPAEHRVRALKALGYLERGEEALDSPGKPGGQG >tr|Q721S0|021S0_THET2 DNA helicase OS =Thermus thermophilus (strain HB27/ATCC BAA-I63/DSM7039) GN = uvrD PE =4 SV = 1  (SEQ ID NO: 107)MSDALLAPLNEAQRQAVLHFEGPALVVAGAGSGKTRIVVHRVAYLVARRGVEPSEILAVTFINKAAEEMRERLRGLVPGAGEVWVSTFHAAALRILRVYGERVGLRPGFVVYDEDDQTALLKEVLKELALSARPGPIKALLDRAKNRGVGLKALLGELPEYYAGLSRGRLGDVIVRYQEALKAQGALDFGDILLYALRLLEEDEEVLRLVRKRARFIHVDEYQDTSPVQYRFTRLLAGEEANLMAVGDPDQGIYSFRAADIKNILDFTRDYPEARVYRLEENYRSTEAILRFANAVIVKNALRIEKALRPVKRGGEPVRIYRAEDAREFAREVAEEIARIGPPWDRYAVLYRTNAQSRLLEQALAGRGIPARVVGGVGFFERAEVKDLLAYARLALNPLDAVSLKRVLNIPPRGIGPATWARVQLLAQEKGLPPWEALKEAARTFPRPEPLRHEVALVEELQDLVEGPAEAFFRHLLEATDYPAYLREAYPEDAEDRLENVEELLRAAKEAEDLQDFLDRVALTAKAEEPAEAEGRVALMTLHNAKGIEFPVVELVGVEEGLLPHRNEVSTLEGLEEERRLFYVGITRAQERLYLSHAEEREVYGRREPARPSRFLEEVEEGLYEVYDPYRRPPSPPPHRPRPGAFRGGERVVHPRFGPGTVVAAQGDEVIVEFEGFGLKRLSLKYAELKPA >tr|F2NK78|F2NK78_MARHT DNA helicase OS =Marinithermus hydrothermalis (strain DSM14884/JCM11576/TI) GN =Marky_1312 PE = 4 SV = 1 (SEQ ID NO: 108)MDLLRDLNPAQREAVQHYTGPALVVAGAGSGKTRTVVERIAYLIRHRGVIPTEILAVTFTNKAAGEMKERLARMVGPAARELWVSTFHSAALRILRVIGEYIGLKPGFVVYDEDDQLALLKEVIGGLGLETRPQYARGVIDRIKNRMWSVDAFLREAEDWVGGLPKEQMAAVYQAYEARMRALGAVDMDTDLKVIGLFEABPEVLHRVQQRARFIHVDEYQDINPAQYRLTRLLAGAERNLMVVGDPDQSIYGFRNADIHNILNFEKDYPDARVYRLEENYRSTEAILRVANAVIERNALRLEKTLRPVRSGGDPVFLYRAPDHREEAAFVAREVQRLKGRGRRLDEAVLYRTNAQSRVLEEAFRRQNLGVRIVGGVGFYERREVKDVLAYARAAVNPADDLAVKRVLNVPARGIGQTSLARDSQLAETARVSFFEALRRAGEVLARPQAQAVQRFVALIEGLANAAYDIGPDAFIRLVLAEIGYADMLRREPDGEARLENLEELLRAAREINEEQHAGIIADFLDEVALTARAEEPEGEVPAEAVILMILHNARGLEETVVFIVGVEEGLLPHRSSTARVEDLEEERRLFYVGIRAQERLYLILSEEREIYGRREAVRASREILEDIPEAFLQPLSPFGEPLGAGREPVAVRPTRRSSAAGGFRGGEKVRHPRFGQGLVVAASGEGDRQEVIVEIFAGVGLKKLLVKYAGLERIEL

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All patents, patent applications, patent application publications andother publications that are cited herein are hereby incorporated byreference as if set forth in their entirety.

It should be understood that the methods, procedures, operations,composition, and systems illustrated in figures may be modified withoutdeparting from the spirit of the present disclosure. For example, thesemethods, procedures, operations, devices and systems may comprise moreor fewer steps or components than appear herein, and these steps orcomponents may be combined with one another, in part or in whole.

Furthermore, the present disclosure is not to be limited in terms of theparticular embodiments described in this application, which are intendedas illustrations of various embodiments. Many modifications andvariations can be made without departing from its scope and spirit.Functionally equivalent methods and apparatuses within the scope of thedisclosure, in addition to those enumerated herein, will be apparent tothose skilled in the art based on the foregoing descriptions.

What is claimed is:
 1. A modified helicase comprising a first subdomaincomprising a 1A or 1B subdomain having a first amino acid and a secondsubdomain comprising a 2B subdomain having a second amino acid, whereinsaid first amino acid is less than about 20 Å from said second aminoacid when the helicase is in an active conformation, wherein the firstamino acid corresponds to any one of positions 84-116 or 178-196 of thehelicase amino acid sequence, relative to SEQ ID NO:32; wherein thesecond amino acid corresponds to any one of positions 388-411, 422-444,and 518-540 of the helicase amino acid sequence, relative to SEQ IDNO:32; wherein a side chain of the first amino acid is covalentlycrosslinked to a side chain of the second amino acid with a linker toform an active, conformationally-constrained helicase; wherein theconformationally-constrained helicase comprises at least one degree offreedom less than a helicase that is not constrained as such; whereinsaid helicase is selected from the group consisting of a Rep helicasefrom E. coli, a UvrD helicase from E. coli, a PcrA helicase from B.stearothermophilus, or a homolog thereof; and wherein theconformationally-constrained helicase enhances an unwinding function ofthe helicase.
 2. The modified helicase of claim 1, wherein said firstsubdomain and said second subdomain comprise no more than a total of twocysteine residues.
 3. The modified helicase of claim 1, wherein thefirst amino acid is covalently crosslinked to the second amino acid by achemical crosslinker.
 4. A modified Rep helicase or homolog thereofcomprising an amino acid at position 178 covalently crosslinked to anamino acid at position 400, relative to SEQ ID NO:32, to form an active,conformationally-constrained Rep helicase or homolog thereof.
 5. A kitfor performing helicase dependent amplification, comprising: theconformationally-constrained helicase of claim 1; and amplificationreagents.
 6. A modified E. coli Rep helicase or homolog thereofcomprising: a first subdomain having a first amino acid, a secondsubdomain having a second amino acid, and an axis vector defined by thealpha carbon of ILE371 from which the vector originates and the alphacarbon of SER280 or the alpha carbon of ALA603, wherein theta is anangle of rotation of said first amino acid and said second amino acidaround the axis vector, wherein a theta between said first amino acidand said second amino acid is between about 355 degrees and about 25degrees when the helicase is in an active conformation, wherein a sidechain of the first amino acid is covalently crosslinked to a side chainof the second amino acid with a linker to form an active,conformationally-constrained helicase; wherein theconformationally-constrained helicase comprises at least one degree offreedom less than a helicase that is not constrained; and wherein theconformationally-constrained helicase enhances an unwinding function ofthe helicase.
 7. The modified E. coli Rep helicase or homolog thereof ofclaim 6, wherein the first amino acid comprises a mutation at any one ofpositions 84-116 or 178-196 of the helicase amino acid sequence,relative to SEQ ID NO:32, to form an active,conformationally-constrained Rep helicase or homolog thereof.
 8. Themodified E. coli Rep helicase or homolog thereof of claim 6, wherein thesecond amino acid comprises a mutation at any one of positions 388-411,422-444, and 518-540 of the helicase amino acid sequence, relative toSEQ ID NO:32, to form an active, conformationally-constrained Rephelicase or homolog thereof.
 9. The modified helicase of claim 1 whereinthe first amino acid and the second amino acid comprise an unnaturalamino acid or a natural amino acid.
 10. The modified helicase of claim 1comprising a cysteine or homocysteine.
 11. A modified helicasecomprising a first subdomain comprising a 1A or 1B subdomain having afirst amino acid and a second subdomain comprising a 2B subdomain havinga second amino acid, wherein said first amino acid is less than about 20Å from said second amino acid when the helicase is in an activeconformation, wherein the first amino acid corresponds to any one ofpositions 84-116 or 178-196 of the helicase amino acid sequence,relative to SEQ ID NO:32; wherein the second amino acid corresponds toany one of positions 388-411, 422-444, and 518-540 of the helicase aminoacid sequence, relative to SEQ ID NO:32; wherein a side chain of thefirst amino acid is covalently crosslinked to a side chain of the secondamino acid with a linker to form an active, conformationally-constrainedhelicase; wherein the conformationally-constrained helicase comprises atleast one degree of freedom less than a helicase that is not constrainedas such; wherein said helicase is selected from the group consisting ofa Rep helicase, a UvrD helicase, a PcrA helicase, or a homolog thereof;wherein the conformationally-constrained helicase enhances an unwindingfunction of the helicase; and wherein said helicase comprises a sequenceselected from the group consisting of SEQ ID NOs:4 and
 12. 12. Themodified helicase of claim 1 wherein the first amino acid is covalentlycrosslinked to the second amino acid by a disulfide bond or abis-maleimide crosslinker.
 13. The modified helicase of claim 1 whereinthe first amino acid is covalently crosslinked to the second amino acidby a chemical crosslinker having a length of from about 6 Å to about 25Å.
 14. The modified helicase of claim 1 wherein the first amino acid iscovalently crosslinked to the second amino acid by a chemicalcrosslinker selected from the group consisting of:

1-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione,

1-[6-(2,5-dioxopyrrol-1-yl)hexyl]pyrrole-2,5-dione,

1-[2-[2-(2,5-dioxopyrrol-1-yl)ethyldisulfanyl]ethyl]pyrrole-2,5-dione,

1-[2-(2,5-dioxopyrrol-1-yl)phenyl]pyrrole-2,5-dione,

N,N′-bis[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethyl]-N,N′-diphenylbutanediamide,and

1-[2-(2,5-dioxopyrrol-1-yl)ethyl]pyrrole-2,5-dione.